Enzyme preparations yielding a clean taste

ABSTRACT

The present invention describes a intracellular produced lactase, which comprises less than 40 units arylsulfatase activity per NLU of lactase activity. The invention also provides a process comprising treating a substrate with an enzyme preparation, wherein the enzyme preparation is substantially free from arylsulfatase.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.12/094,541, filed May 21, 2008, which is a 371 of PCT/EP2006/68979,filed 28 Nov. 2006, which claims priority to EP 05111392.6, filed 28Nov. 2005 and EP 06113062.1, filed 25 Apr. 2006, the contents of whichare incorporated herein by reference in their entireties.

FIELD OF THE INVENTION

The invention relates to a process for treating a substrate with anenzyme preparation, to a new enzyme preparation and a process forpreparing an enzyme preparation. The invention also relates to lactase.

BACKGROUND OF THE INVENTION

The use of enzymes to improve the chemical, physico-chemical ororganoleptic nature of food grade products is wide spread. Also in theprocessing of cow milk and other animal derived substrates, the use ofenzymes adds significant value to the end product. Examples areincubations with lactase to render milk acceptable for lactoseintolerant individuals, proteolytic hydrolysis of casein and wheyproteins to alleviate allergenicities and to improve foamcharacteristics, the modification of egg phospholipids usingphospholipase A2 to improve baking performance and stabilize mayonaises,the use of transglutaminase on meat and fish products to improvehardness and elasticity as well as the removal of oxygen from eggproducts or grated cheese by adding glucose oxidase. Additionally,enzyme treatments are being used to enhance the flavor of various animalderived food products. For example, proteases are being used to speed upflavor development in fish and meat extracts. Furthermore, acceleratingflavor development in cheese is a well known target. Whereas EMC (EnzymeModified Cheese) is an established product in which primarily variouslipases are used, speeding up the subtle taste changes involved in theaging of cheeses by adding minor quantities of exoproteases, lipases oresterases, is a more recent development.

The invention also relates to lactase. Lactase or, β-galactosidase (E.C:3.2.1.23) is an enzyme, which catalyzes the hydrolysis of lactose (adisaccharide) into its component monosaccharides glucose and galactose.Lactose is present in dairy products and more specifically in milk,skimmed milk, cream and other milk products. The breakdown of lactoseoccurs in the intestinal wall of the human body (and other mammals) bythe natural presence of lactase.

The nutritional and functional problems caused by lactose in mostpopulations that lack lactase are well known and described. Members ofsuch populations cannot hydrolyze the lactose, which in such casespasses into the large intestine, where it produces dehydration, poorcalcium absorption, diarrhoea, flatulence, belching and cramps, and, insevere cases, even watery explosive diarrhoea.

An important industrial application of lactase is in the production oflactose-hydrolyzed milk products for lactose intolerant individuals.Such hydrolysed milk products include pasteurized milk, UHT-milk andmilk reconstituted from all or part of its original constituents with orwithout intermediate processing steps such as protein hydrolysis.Treatment with lactase may be done prior to and after the heat-treatmentof the milk. The lactase treatment may be done by adding the enzyme tothe milk. The solubility properties of lactose are such that it may leadto its crystallization, leading to a sandy or gritty texture. Suchundesired texture may be found in some dairy products such as condensedmilk, evaporated milk, dry milk, frozen milk, ice cream, and inconfectionary products with a high content of milk. Full or partialhydrolysis of lactose by lactase eliminates this problem, providingproducts with a homogeneous texture and as a result a higher consumeracceptance.

Another industrial application of lactase is to increase sweet taste inlactose containing products like milk or yoghurt. The hydrolysis oflactose in such products results in increased sweet taste as a result ofthe production of glucose. Another industrial application of lactase isthe hydrolysis of lactose products containing dairy components such asbread. Lactose is added in such products to enhance flavour, retainmoisture, provide browning and improve toasting properties. Hydrolyzedlactose syrups are promising in terms of e.g. enhancing crust-colourdevelopment, improving flavour and aroma, modifying texture, extendingshelf life and strengthening loaf structure.

Lactose hydrolysis by lactase in fermented milk products such as yoghurtwill increase sweet taste. However, when the lactase is added prior tothe beginning of the fermentative process, it may increase the rate ofacid development and thus reduce processing times. The lactase treatmentof milk or milk-derived products such as whey makes such productssuitable for application in animal feed and pet food for lactoseintolerant animals such as cats. The lactose hydrolysis allows themanufacture of a higher concentrated whey and at the same time preventsgut problems, similar to those described earlier for lactose-deficientpatients. Lactose hydrolyzed whey is concentrated to produce a syrupcontaining 70-75% solids and is used as a food ingredient in ice cream,bakery and confectionary products.

Lactases have been described for and isolated from a large variety ororganisms, including micro-organisms. Lactase is often an intracellularcomponent of micro-organisms like Kluyveromyces and Bacillus.Kluyveromyces and especially K. fragilis and K. lactis, and other yeastssuch as those of the genera Candida, Torula and Torulopsis are a commonsource of yeast enzymes lactases, whereas B. coagulans or B circulansare well known sources for bacterial lactases. Several commerciallactase preparations, derived from these organisms are available such asMaxilact® (from K. lactis, produced by DSM, Deflt, The Netherlands). Allthese lactases are so called neutral lactases since they have a pHoptimum between pH=6 and pH=8. Several organisms such as Aspergillusniger and Aspergillus oryzae can produce extracellular lactase, and U.S.Pat. No. 5,736,374 describes an example of such lactase, produced byAspergilllus oryzae. The enzymatic properties of lactases like pH- andtemperature optimum vary between species. In general, however, lactasesthat are excreted show a lower pH-optimum of pH=3.5 to pH=5.0;intracellular lactases usually show a higher pH optimum in the region ofpH=6.0 to pH=7.5, but exceptions on these general rules occur. Thechoice for a neutral or acidic lactase depends on the pH profile in theapplication. In applications with neutral pH, neutral lactases areusually preferred; such applications include milk, ice cream, whey,cheese, yoghurt, milk powder etc. Acid lactases are more suited forapplications in the acidic range. The appropriate lactase concentrationis dependent on the initial lactose concentration, the required degreeof hydrolysis, pH, temperature and time of hydrolysis.

Although aimed at improving the functionality and/or taste profiles ofthe food product, occasionally an enzyme treatment can have unexpectedand undesirable side effects. An example of an undesirable side effectis the development of off-flavor as a result of the enzyme treatment.

Mettall et. al, The Australian Journal of Dairy Technology, (1991),46-48 describes the problem of off-flavor development when milk istreated with lactase. According to this publication high levels ofprotease will result in the rapid development of off-flavors. Productionprocesses are therefore optimised to minimize proteolytic sideactivities in order to reduce the risk of off-flavour formation. Anexample of a purification process for K lactis derived lactase isdescribed in WO 02/081673.

It is found that even lactase preparations with low protease activitycan still give rise to off-flavour formation. This is especially thecase for the neutral lactases, derived from the cytoplasm of yeast. Theoff-flavour formation that is associated with the use of lactasepreparations is especially critical for lactose hydrolysed UHT-milk. Thelactases that are used in this case are neutral lactases because oftheir favourable pH optimum for milk. The UHT milk has received a highheat treatment to obtain a shelf life of several months at roomtemperature. The long storage times outside the refrigerator make theseproducts especially prone to off-flavour formation: even a very lowoff-flavour formation rate can give rise to significant off-flavourformation after several months of storage, making the productunattractive for consumption.

SUMMARY OF THE INVENTION

Surprisingly it is now found that the presence of arylsulfatase ascontaminating side activity in enzyme preparations, even at very lowlevels, can lead to a strong development of off-flavor in a product whena substrate is treated with the preparation, and that the use of anenzyme preparation having no or a reduced aryl sulfatase activityresults in a strong reduction of off-flavor development.

Accordingly, the invention provides a process, in one aspect, a processcomprising treating a substrate with an enzyme preparation, wherein theenzyme preparation is substantially free from arylsulfatase.

The invention also provides, in further aspects, enzyme preparationssubstantially free from arylsulfatase.

The invention also provides, in a particular aspect, lactase whichcomprises less than 40 units arylsulfatase activity per NLU of lactaseactivity.

The lactase preparation according to the invention may advantageously beused in food and feed products to hydrolyse lactose without theformation of off-flavour compounds.

We have surprisingly found that aryl-sulfatase is a crucial enzymeactivity, responsible for off-flavor formation. We have foundconfirmative evidence by adding aryl-sulfatase to UHT-milk and whichresulted in that this single enzyme is capable to mimic the off-flavouroften observed in lactase-treated UHT-milk.

Without wishing to be bound by any scientific theory, it is believedthat hydrolysis of metabolic conjugates, in particular alkyl phenolssubstituted with a sulfate group, by arylsulfatases is a mechanismresulting in the development of off-flavor. Accordingly, the enzymepreparations according to the invention are particular advantageous forthe treatment of substrates containing an alkyl phenol substituted witha sulphate group.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect, the invention provides a lactase which comprises lessthan 40 units arylsulfatase activity per NLU of lactase activity.Preferably, the lactase comprises less than 30 units arylsulfataseactivity per NLU of lactase activity, more preferably less than 20 unitsarylsulfatase activity per NLU of lactase activity and most preferablyless than 10 units arylsulfatase activity per NLU of lactase activity.The aryl-sulfatase units are defined in example 2 and are normalized forlactase activity expressed in NLU and also defined in example 2.

The lactase may be an intracellular or an extracellular producedlactase. In a preferred embodiment, the lactase is intracellularproduced lactase.

In a preferred embodiment, the lactase is a neutral lactase. The neutrallactase may have a pH optimum between pH=6 and pH=8.

Neutral lactase preparations are usually derived from the cytoplasm ofmicro-organisms. Their production includes the (large scale)fermentation of the micro-organism, followed by isolation of thelactase. The latter requires the disruption of the cell wall in order torelease the enzyme from the cytoplasm. Several techniques can be used toobtain cell lysis, including permeabilization of the cell wall byorganic solvents such as octanol, sonication or French Pressing. Otherenzymes beside lactase are released at the same time from the cytoplasm,including proteases.

In a preferred embodiment, the lactase has less than 0.5 RFU/minprotease activity per NLU of lactase activity.

The intracellular lactases which can be purified according to thepresent invention have been described for and isolated from a largevariety or organisms, including microorganisms. Lactase is often anintracellular component of micro-organisms like Kluyveromyces andBacillus. Kluyveromyces and especially K. lactis, K marxinus and K.fragilis, and other yeasts such as those of the genera Candida, Torulaand Torulopsis are a common source of yeast enzymes lactases, whereas B.coagulans or B circulans are well known sources for bacterial lactases.Several commercial lactase preparations, derived from these organismsare available such as Maxilact® (from K. lactis, produced by DSM). Allthese lactases are so called neutral lactases since they have a pHoptimum between pH=6 and pH=8.

Intracellular lactases have been described for various species, and forseveral of them their amino acid sequences and/or their DNA sequencesare known. The sequence information is publicly available in sequencedatabases, for example in GenBank (Bethesda, Md. USA), EuropeanMolecular Biology Laboratory's European Bioinformatics Institute(EMBL-Bank in Hinxton, UK), the DNA Data Bank of Japan (Mishima, Japan)and the Swissprot (Switzerland). Lactases can be identified in genomesbased on homology in either the gene and/or protein sequences. Crudepreparations of intracellular enzymes are characterized by the presenceof several enzymes only occurring in the cytoplasm of the cell, such asthe enzymes involved in the central metabolism of the cell, includingthose involved in glycolysis.

Extracellular lactases have also been described. They are generallyrecognized as extracellular enzymes because they contain a peptidesequence called leader sequence. This leader sequence is recognized insome way by the cell that produces the enzymes as a signal that theenzyme should be exported out of the cell. During secretion, the leadersequence is usually removed. Extracellular lactases have been describedfor various species, e.g. Aspergillus oryzae. Crude preparations ofextracellular lactases are characterized by the absence of intracellularenzymes and the presence of typical extracellular enzymes likeproteases. The type of extracellular enzymes found varies with theorganism and are typical for that organism. Due to cell lysis duringfermentation or processing, low levels of intracellular enzymes can befound in such extracellular enzyme preparations.

Lactase enzymes can thus be classified as extracellular or intracellularbased on comparison of their amino acid sequence with those of otherknown lactases. In principle, an intracellular lactase can be providedwith a leader sequence. This could result in excretion of the lactasefrom the cell into the medium. Crude preparations of such enzymes wouldbe characterized by a lactase, classified as intracellular on basis ofits amino acid sequence, in the presence of typical extra-cellularenzymes and absence or low levels of typical intracellular enzymes.

Preferred intracellular lactases used in the present invention are: K.lactis lactase having an amino acid sequence as described inhttp://www.ebi.uniprot.orq/entry/BGAL_KLULA or a lactase having an aminoacid sequence which is at least 90%, preferably at least 95% identicalwith the amino acid sequence of K. lactis. K. marxianus lactase havingan amino acid sequence as described inhttp://www.ebi.uniprot.orq/entry/Q6QTF4_KLUMA or a lactase having anamino acid sequence which is at least 90%, preferably at least 95%identical with the amino acid sequence of K. lactis. B. circulanslactase having an amino acid sequence as described inhttp://www.ebi.uniprot.orq/uniprot-srv/uniProtView.do?proteinld=031341_BACCI&pager.offset=0http://www.ebi.uniprot.orq/uniprot-srv/uniProtView.do?proteinld=Q45092_BACCI&paqer.offset=0http://www.ebi.uniprot.orq/uniprot-srv/uniProtView.do?proteinld=Q45093_BACCI&pager.offset=0or a lactase having an amino acid sequence which is at least 90%,preferably at least 95% identical with the amino acid sequence of B.circulans.

The terms “homology” or “percent identity” are used interchangeablyherein. It is defined here that in order to determine the percentidentity of two amino acid sequences or of two nucleic acid sequences,the sequences are aligned for optimal comparison purposes (e.g., gapscan be introduced in the sequence of a first amino acid or nucleic acidsequence for optimal alignment with a second amino or nucleic acidsequence). The amino acid residues or nucleotides at corresponding aminoacid positions or nucleotide positions are then compared. When aposition in the first sequence is occupied by the same amino acidresidue or nucleotide as the corresponding position in the secondsequence, then the molecules are identical at that position. The percentidentity between the two sequences is a function of the number ofidentical positions shared by the sequences (i.e., % identity=number ofidentical positions/total number of positions (i.e. overlappingpositions)×100). Preferably, the two sequences are the same length. Theskilled person will be aware of the fact that several different computerprograms are available to determine the homology between two sequences.For instance, a comparison of sequences and determination of percentidentity between two sequences can be accomplished using a mathematicalalgorithm. In a preferred embodiment, the percent identity between twoamino acid sequences is determined using the Needleman and Wunsch (J.Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporatedinto the GAP program in the GCG software package (available athttp://www.qcq.com), using either a Blossom 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6. The skilled person will appreciate thatall these different parameters will yield slightly different results butthat the overall percentage identity of two sequences is notsignificantly altered when using different algorithms.

The preparation of intracellular lactases requires the disruption of thecells to release the lactase enzyme. At the same time, other cytoplasmicenzymes are released. The quality of an industrial preparation of thelactase is determined by ratio of side activities to lactase activity.Especially proteases are critical side enzymes since they are known tolead to unwanted side effects in application, such as milk clotting oroff-flavour formation in milk. Off-flavour formation is especiallycritical in products with a long shelf life and which are stored at roomtemperatures. One such product is UHT-milk, and off-flavour formation isa known problem for lactose hydrolysed UHT-milk. The UHT-milk is verysensitive to off-flavour formation; when a lactase preparation does notgenerate off-flavour in UHT-milk, it will usually also not generateoff-flavour in other applications. Compounds associated with off-flavourformation in milk, and especially UHT-milk, are related to bothproteolysis and Maillard reactions (Valero et al (2001) Food Chem. 72,51-58). Any proteases present as side activities in lactase preparationspotentially enhance the off-flavour formation; it is unclear what levelsof proteases are required, but with storage times of several months evenvery low proteolytic activity could be important. The UHT-milk is verysensitive to off-flavour formation; when a lactase preparation does notgenerate off-flavour in UHT-milk, other than the off-flavours described(as e.g. described in Valero et al (2001) Food Chem. 72, 51-58) it willusually also not generate off-flavour in other applications. TheUHT-application is therefore a good method to evaluate the quality oflactase preparations regarding their off-flavour potential. Sinceproteases were held at least partly responsible for the off-flavourformation, efforts have focussed on reducing protease levels of lactaseproducts. We have found, however, that a reduction of protease levelsdoes not lead to complete removal of off-flavour formation in UHT-milk.We have surprisingly found that aryl-sulfatase is a crucial enzymeactivity, responsible for off-flavor formation. We have foundconfirmative evidence by adding aryl-sulfatase to UHT-milk and whichresulted in that this single enzyme is capable to mimic the off-flavouroften observed in lactase-treated UHT-milk.

According to the present invention a chromatographic process isdisclosed to remove the aryl-sulfatase from the lactase enzyme, which ispreferably derived from K lactis.

We performed a detailed sensory analysis of various samples of UHT-milkthat either contained no off-flavour or that contained significantlevels of off-flavour (example 1). These sensory analyses were combinedwith detailed analysis of the chemical composition of the samples.Several compounds were identified as key aroma compounds, and most ofthem had been described previously as associated with UHT-milk.Surprisingly, p-cresol was also identified as a key off-flavourcompound. This compounds has not been described previously among theoff-flavour compounds in UHT-milk (Valero et al (2001) Food Chem. 72,51-58). It can be generated by aryl-sulfatase from its sulfate conjugatethat is present is very low amounts (ppb-levels) in milk (V. Lopez, R.C. Lindsay J Agric. Food Chem. (1993), 41, 446-454; M. Killic & R. C.Lindsay, J Dairy Sci (2005) 88, 7-12; M Kilic & R. C. Lindsay J AgricFood Chem (2005) 53, 1707-1712). We have surprisingly found thataryl-sulfatase is an enzyme activity in lactase preparations andresponsible for off-flavour formation. We confirmed this by addingaryl-sulfatase to UHT milk and found that this single enzyme is indeedcapable to mimic the off-flavour often observed in lactase treatedUHT-milk. We subsequently developed a chromatographic process to removethe aryl-sulfatase from the lactase enzyme, which is derived from K.lactis. We found that the removal of aryl-sulfatase also results inremoval of off-flavour formation in UHT-milk, as concluded from trialswith taste panels. The aryl-sulfatase levels in the final lactaseproduct are <20 units aryl-sulfatase, preferably <10 unitsaryl-sulfatase, even more preferably <8 units aryl-sulfatase and mostpreferably 0 units aryl-sulfatase. The aryl-sulfatase units are definedin example 2 and are normalized for lactase activity expressed in NLUand also defined in example 2). Several purifications routes forlactases have been described (e.g. in WO02/081673), but thesepurification processes were not directed to remove the aryl-sulfatase.The present results show that aryl-sulfatase and lactase, both derivedfrom K lactis, have a very similar elution behaviour on ion exchange(Q-sepharose) and hydrophobic interaction (butyl-sepharose)chromatography. Therefore it is expected that the described prior artroutes will not result in lactase preparations free from aryl-sulfataseactivity.

Beside the reduction of aryl-sulfatase levels in lactase preparations bychromatography there are other ways to reduce or eliminatearyl-sulfatase activity from the lactase preparation. These are 1) theaddition of sulfate to the growth medium. Sulfate is known the repressaryl-sulfatase expression (Beil et al. (1995) Eur. J. Biochem. 229,385-394), and sulfate addition to the medium is therefore expected tolower aryl-sulfatase levels; 2) elimination or disruption of the genefor aryl-sulfatase from the genome of the organism by either randommutagenesis techniques or by a directed approach using e.g. molecularbiology technologies known to the person skilled in the art, 3)screening and selection of a strain that is a natural low producer ornon-producer of aryl-sulfatase activity; 4) addition of an inhibitor ofthe enzyme. It is e.g. known that certain classes of aryl-sulfatases areinhibited by phosphate ions.

Metabolic conjugates such as sulfates, glucuronides and phosphates arepresent in milk from various species, including cows milk (Lopez et al(1993) J Agric Food Chem. 41, 446-454; Killic et al (2005) J Dairy Sci88, 7-12). Metabolic conjugation is a universally accepted means ofdetoxification and enhancement of aqueous solubility of foreignsubstances in mammals. Conjugates are most effectively formed by theliver and kidney, and they circulate in the bloodstream beforeelimination principally in the urine and bile. Conjugates ofalkylphenols and a variety of other compounds have been found in milkfrom e.g. cow, goat and sheep (Lopez et al (1993) J Agric Food Chem. 41,446-454). The nature and diversity of metabolic conjugates is very wide,and includes conjugates of thiophenols, phenols, o-cresol and p-cresol.The conjugation can result in the attachment of a sulphate-, phosphateor glucoronide groups. These groups can be released from the conjugateby enzymes like aryl-sulphatases, phosphates and glucoronidases,resulting in release of the toxic compound. The presence of severaltypes of conjugates has been demonstrated in milk from cow, sheep andgoat; the relative abundance of the conjugates varies betweenpreparations and is at least partly species related (Lopez et al (1993)J Agric Food Chem. 41, 446-454). In cows milk, sulfate-conjugates weredemonstrated to be the most abundant conjugates, but in sheep milkphosphate-conjugates are more abundant than sulfates (Lopez et al (1993)J Agric Food Chem. 41, 446-454).

In the present application it is demonstrated that the conjugates thatare present in milk are the substrate for side activities in neutrallactase preparations. It is known that the concentration levels of theseconjugates may vary for a species over time (Kilic et al, (2005) J dairySci 88, 7-12) and between species (Lopez et al (1993) J Agric Food Chem41, 446-454). It is anticipated that this may affect the requirementsfor the lactase preparation. For instance it is anticipated that forsheep milk, in which phosphate-conjugates are very abundant, thetolerance for phosphatase-levels in lactase preparations is much lowercompared to the situation where the same lactase preparation is used incows milk which has very low levels of phosphate-conjugates. In thisrespect, there is no difference between preparation of intracellularlactase or extracellular lactase preparations.

In a further aspect, the invention provides a process for treating asubstrate with an enzyme preparation. The enzyme preparation ispreferably substantially free from aryl sulfatase.

As used herein, an enzyme preparation substantially free fromarylsulfatase may encompass any enzyme preparation, in which thearylsulfatase activity is not present or present at a sufficiently lowlevel that, upon effective dosage of the intended enzyme activity in therelevant production process, no observable decomposition of sulphatedalkylphenols with the associated negative organoleptic effects asdescribed above occurs in said production process.

As used herein, an enzyme preparation substantially free fromarylsulfatase may encompass an enzyme preparation wherein the ratio ofthe arylsulfatase activity divided by the activity of the enzyme ofinterest is below a specified value. Preferred ratio's may varydepending on the enzyme and application used.

By arylsulfatase activity is meant the sulphuric ester hydrolaseactivity able to cleave a phenol sulfate into the phenol and sulfatemoiety as described for EC 3.1.6.1. Definition for the arylsulfataseunit is provided in the Materials & Methods section (and example 2) ofthe present application. Definitions for the activities of the otherenzymes can also be found in the Materials & Methods section of thepresent application.

In a further aspect of the invention, the invention provides an enzymepreparation comprising a carboxypeptidase, which enzyme preparationcomprises less than 10000 units (ASU) of aryl sulfatase activity perunit of carboxypeptidase (CPG). Preferably, the enzyme preparationcomprises less than 5000 units, more preferably less than 1000 units,more preferably less than 500 units, more preferably less than 100units, more preferably less than 50 units, more preferably less than 10units of arylsulfatase activity per carboxypeptidase unit (CPG).

In a further aspect, the invention provides an enzyme preparationcomprising a proline-specific protease, which enzyme preparationcomprises less than 300*10E3 units (ASU) of arylsulfatase activity perunit of prolin protease (PPU). Preferably, the enzyme preparationcomprises less than 100*10E3 units, preferably less than 50*10E3 units,preferably less than 10*0E3 units, preferably less than 5000 units ofarylsulfatase per protease unit (PPU).

In a further aspect, the invention provides an enzyme preparationcomprising a (neutral) lactase, which enzyme preparation comprises lessthan 40 units (ASU) arylsulfatase activity per NLU of lactase activity.Preferably, the enzyme preparation comprises less than 30 unitsarylsulfatase activity per NLU of lactase activity, more preferably lessthan 20 units arylsulfatase activity per NLU of lactase activity andmost preferably less than 10 units arylsulfatase activity per NLU oflactase activity.

In a further aspect, the invention provides an enzyme preparationcomprising an (acid) lactase, which enzyme preparation comprises lessthan 400 units (ASU) of arylsulfatase activity per ALU of lactaseactivity. Preferably, the enzyme preparation comprises less than 100units (ASU) of arylsulfatase activity, preferably 30 units arylsulfataseactivity per ALU of lactase activity, more preferably less than 20 unitsarylsulfatase activity per ALU of lactase activity and most preferablyless than 10 units arylsulfatase activity per ALU of lactase activity.

In a further aspect, the invention provides an enzyme preparationcomprising an aminopeptidase, which enzyme preparation comprises lessthan 1000 units (ASU) of aryl sulfatase activity per APU, preferablyless than 300 units (ASU) of aryl sulfatase activity per APU, preferablyless than 100 units (ASU) of aryl sulfatase activity per APU, preferablyless than 30 units (ASU) of aryl sulfatase activity per APU, preferablyless than 10 units (ASU) of aryl sulfatase activity per APU.

In a further aspect, the invention provides an enzyme preparationcomprising an esterase and/or a lipase, which enzyme preparationcomprises less than 10*10E6 units (ASU) of aryl sulfatase activity perBGE, preferably less than 3*10E6 units (ASU) of aryl sulfatase activityper BGE, preferably less than 1*10E6 units (ASU) of aryl sulfataseactivity per BGE, preferably less than 300*10E3 units (ASU) of arylsulfatase activity per BGE.

Treatment of a substrate with an enzyme preparation substantially freefrom arylsulfatase may also encompass the treatment of a substratewherein the level of arylsulfatase in the substrate during said treatingis below a specified value.

In a further aspect, the invention provides a process for treating asubstrate with an enzyme preparation, wherein the level of arylsulfatasein the substrate during said treating is at most 500*10E3 arylsulfataseunits per liter of substrate, preferably at most 250*10E3, preferably atmost 100*10E3, preferably at most 50*10E3, preferably at most 25*10E3arylsulfatase units per liter of substrate. Maintaining the level ofarylsulfatase below the abovementioned values was found to be particularadvantageous when the substrate is milk, preferably cow milk.

An enzyme preparation substantially free from aryl sulfatase may alsoencompass any enzyme preparation obtained by purifying a crude enzymepreparation which contains an enzyme of interest and arylsulfatase,wherein arylsulfatase is separated from the enzyme of interest.

Accordingly, the invention also provides a process for preparing anenzyme preparation, which process comprises purifying a crude enzymepreparation which contains an enzyme of interest and arylsulfatase,wherein arylsulfatase is separated from the enzyme of interest. Theprocess may advantageously comprise treating a substrate with thepurified enzyme preparation.

The purification step has the effect that the activity of arylsulfataserelative to the activity of enzyme of interest is reduced. Preferably,the purifying results in a reduction of arylsulfatase activity of atleast 50%, preferably at least 80%, more preferably at least 90%, morepreferably at least 95%, more preferably at least 99%. The skilledperson will appreciate that this is understood to mean that preferably(a_(AS,pur)/a_(enz,pur))/(a_(AS, crude)/a_(enz,crude)) 0.5, preferably0.2, preferably 0.1, preferably 0.05, preferably 0.01, wherein

a_(AS,pur)=arylsulfatase activity in purified enzyme preparation(unit/ml)a_(enz,pur)=activity of enzyme of interest in purified enzymepreparation (unit/ml)a_(AS, crude)=aryl sulfatase activity in crude enzyme preparation(unit/ml)a_(enz, crude)=activity of enzyme of interest in crude enzymepreparation (unit/ml)

The purification can be effected in any suitable manner. In a preferredembodiment, the purifying is by chromatography. Processes for purifyingenzyme preparations using chromatography are known per se. Selecting themost appropriate chromatographic separation methods depend on molecularcharacteristics of both the relevant enzyme and of the relevantarylsulfatase activity present. Relevant molecular characteristics arethe isoelectric point, hydrophobicity, molecular surface chargedistribution, molecular weight of the relevant enzyme and the sideactivity as well as several other protein chemical properties. Apractical background on the use of these characteristics in selectingthe appropriate chromatographic separation process, can be found in a.o.the Protein Purification Handbook (issued by Amersham Pharmacia Biotech,nowadays GE Healthcare Bio-Sciences, Diegem, Belgium). Suitablechromatrographic separation methods comprise ion exchangechromatography, affinity chromatography, size exclusion chromatogrpahy,hydrophobic interaction chromatrography and others. For the presentinvention ion exchange chromatography or hydrophobic interactionchromatography are preferred.

In a preferred embodiment, the purification is performed in a singlechromatographic separation step. The fact that enzymatic activity can beefficiently separated from the contaminating arylsulphatase activity ina single chromatographic step, is particularly advantageous for theindustrial applicability of the process according to the invention.

The enzyme preparation may comprise any suitable enzyme. In a preferredembodiment, the enzyme, hereinafter also referred to as enzyme ofinterest, is a lactase, a protease, a lipase, or an esterase. Enzymesthat may be used according to the invention are disclosed hereinafter.

The internationally recognized schemes for the classification andnomenclature of all enzymes are provided by IUMB. An updated IUMB textfor EC numbers can be found at the internet site:http://vvww.chem.amw/ac.uk/iubmb/enzyme/EC3/4/11/. In this systemenzymes are defined by the fact that they catalyze a single reaction.This implies that several different proteins are all described as thesame enzyme, and a protein that catalyses more than one reaction istreated as more than one enzyme.

According to the system, proteases can be subdivided into endo- andexoproteases. Moreover, socalled di- and tripeptidyl peptidases exist.Endoproteases are those enzymes that hydrolyze internal peptide bonds,exoproteases hydrolyze peptide bonds adjacent to a terminal α-aminogroup (“aminopeptidases”), or a peptide bond between the terminalcarboxyl group and the penultimate amino acid (“carboxypeptidases”). Theendoproteases are divided into sub-subclasses on the basis of catalyticmechanism. There are sub-subclasses of serine endoproteases (EC 3.4.21),cysteine endoproteases (EC 3.4.22), aspartic endoproteases (EC 3.4.23),metalloendoproteases (EC 3.4.24) and threonine endoproteases (EC3.4.25). Proteases typically to clot milk for cheese production, such aschymosin (EC 3.4.23.4) or mucorpepsin (EC 3.4.23.23), all belong to theclass of the aspartic endoproteases.

Among the exoproteases, the so-called aminopeptidases (EC 3.4.11) cansequentially remove single amino-terminal amino acids from protein andpeptide substrates. Among the exoproteases, the carboxypeptidases (EC3.4.16, 3.4.17 and 3.4.18) can sequentially remove singlecarboxy-terminal amino acids from protein and peptide substrates. Di-and tripeptidyl peptidases (EC 3.4.13, 3.4.14 and 3.4.15) can cleave offdipeptides or tripeptides from either the amino- or the carboxyterminalside of peptides or proteins.

In an embodiment of the invention, the enzyme is a protease with theexclusion of an aspartic endoprotease (EC3.4.23).

Other enzymes acting on proteins or peptides and of particular relevancewithin the scope of the present application, are the omega peptidases(EC 3.4.19) and enzymes able to transform side groups of amino acids.The substrate for such transformation reactions can be free amino acidsor protein- or peptide-bound amino acids. Examples of the latter groupof enzymes are enzymes that can selectively hydrolyse gamma amide groupsof protein bound glutamines, i.e. the peptide-glutaminases (EC 3.5.1.43and 3.5.1.44).

Furthermore enzymes able to crosslink proteins or peptides such astransglutaminase (EC 2.3.2.13) and protein-lysine 6-oxidase (EC1.4.3.13) represent typical examples.

Lactase (EC 3.2.1.23), a microbial beta-galactosidase able to decomposethe lactose, is of particular relevance within the scope of the presentapplication.

Lipases and esterases are of particular relevance within the scope ofthe present application because these enzymes are commonly used in theproduction of EMC's (Enzyme Modified Cheeses) and enjoy an increasinginterest for accelerating cheese aging. Therefore, lipases and esterasesare prime candidates to be purified according to the process of thecurrent invention. According to the IUMB system lipases and esterasesbelong to the carboxylic ester hydrolases (EC 3.1.1). Whereas esterasescan act on a broad variety of substrates, lipases (EC 3.1.1.3) cleavetriacylglycerols only. Lipases capable of removing formate, acetate,propionate or butyrate from triacylglycerols are sometimes also referredto as “esterases”. In the present application the term “esterase” refersto enzymes that can efficiently remove such short chain carboxylic acidsfrom triacylglycerols. The recovery of amphiphilic enzymes such aslipases or esterases is optionally improved by using bile acids oranother food grade emulsifier. Methods for the activity determination oflipases and esterases are provided in the Materials & Methods section.

Industrially available, food grade enzyme preparations are typicallyobtained from mammalian tissue, e.g. trypsin from pancreas, or fromplant material, e.g. papain from papaya fruits. In a preferredembodiment the enzyme is obtained from a microbial strain, for instancebacteria, e.g. Bacillus species, or yeasts, e.g. Saccharomyces,Kluyveromyces or Pichia, or filamentous fungi. Filamentous fungi knownto produce food grade enzyme preparations are for instance Aspergillus,Rhizomucor, Rhizopus, Trichoderma and Talaromyces. In an embodiment ofthe invention, the enzyme preparation is produced by or derived from afilamentous fungus, for instance Aspergillus niger or Aspergillusoryzae. As used herein such enzyme preparations also encompassself-cloned enzyme preparations produced by either A. niger or by A.oryzae.

The enzyme of interest may be produced by microbial fermentationprocesses using fungi that produce and preferably secrete the proteaseof interest in the fermentation broth. In the art, such fermentationprocesses are known, see for example WO 02/45524. In the processes ofthe prior art, the enzyme may be recovered from the fermentation brothby techniques also known in the art. As a first step, the cells of theproduction organism may be separated from the broth by centrifugation orfiltration. The cell free broth may be concentrated, for example byultrafiltration, and subsequently chromatographically purified. Fungalstrains typically produce more than one arylsulphatase activity so thatthe chromatographic separation of the relevant enzyme from thesearylsulfatase activities in a single step, is not trivial. An additionalcomplication is that the different enzyme activities secreted by aspecific microorganism, i.e. the enzyme activity sought as well as thevarious arylsulphatase activities, have isoelectric points closelytogether. Upon chromatographic separation of the desired enzymaticactivity and the contaminating arylsulfatase activities, the purifiedenzyme preparation thus obtained may be stabilized.

In case the enzyme is not secreted by the microorganism but remainsintracellular, the production organism may be recovered by filtration orcentrifugation after which the retained cells may be lysed to releasethe relevant enzymatic activity. After another filtration orcentrifugation step to remove the cell debris, the liquid fraction maybe concentrated and stabilized as described above for the secretedenzyme.

The purified and liquid enzyme preparations may be concentrated andmixed with known stabilizers such as glycerol or other polyols.Alternatively, solid preparations may be obtained from concentratedenzyme solutions by known precipitation and/or evaporation stepsfollowed by well known (spray) drying techniques.

According to the invention a substrate may be treated with the enzymepreparation. The substrate may be any suitable substrate. Preferably,the substrate is a proteinaceous substrate. The proteinaceous substratemay be any substrate comprising protein. In a preferred embodiment, thesubstrate contains milk protein, for instance casein and/or wheyprotein. Examples of preferred substrates are milk, milk-derivedproducts, fermented milk products (for instance yoghurt) whey and/orhydrolysates. The substrate may also comprise meat.

As a hydrolysate may be used any product that is formed by the enzymatichydrolysis of a proteinaceous substrate protein, preferably an animalderived substrate protein. Whey protein hydrolysates, caseinhydrolysates and skim milk hydrolysates are preferred.

In a preferred embodiment, the substrate contains an alkyl phenolsubstituted with a sulfate group. By alkylphenol is meant a phenol groupof which at least one aromatic proton has been replaced by an alkylgroup. The length of the alkyl group may vary and may be branched orsubstituted. Preferred alkylphenols are methyl and ethyl phenols.

By a sulphated alkylphenol is meant an alkylphenol which is conjugatedat the hydroxyl group by sulfation.

Arylsulphatase (EC 3.1.6.1) is a sulphuric ester hydrolase able tocleave an alkyl phenol sulfate into the alkyl phenol and sulfate moiety.

The treatment of the substrate may involve any process wherein asubstrate is contacted with the enzyme preparation. The treatment mayinvolve any process wherein the substrate is incubated in the presenceof the enzyme preparation. The enzyme preparation may be added to thesubstrate in any suitable manner.

The process may be any process wherein a product is produced, forinstance a nutritive product, preferably a dairy product. As usedherein, a dairy product encompasses any composition that contains milkprotein, for instance casein and/or whey protein. Examples are milk,milk-derived products, fermented milk products (e.g. yoghurt), condensedmilk, evaporated milk, dry milk, frozen milk, ice cream, whey; and/orcheese. The product may also be a hydrolysate.

The enzyme preparation may be used to prepare any suitable product, forinstance a nutritive product, preferably a dairy product.

The invention also relates to the use of the enzyme preparationaccording to the invention to prevent or reduce the development ofoff-flavor.

In an aspect, the invention provides a process to produce a host cellwhich is an arylsulfatase deficient strain, which comprises bringing aculture which produces arylsulfatase under conditions that part of theculture is modified to form the host cell which is arylsulfatasedeficient and isolating the host cell.

In a preferred embodiment mutagenesis conditions are used, preferablyrandom mutagenesis conditions such as physical or chemical mutagenesis.

In a preferred embodiment, recombinant genetic manupilation techniquesare used, preferably one-step gene disruption, marker insertion, sitedirected mutagenesis, deletion, RNA interference, anti-sense RNA.

The invention further provides a process to produce a polypeptide by amethod comprising:

(a) cultivating an arylsulfatase deficient host cell in a nutrientmedium, under conditions conductive to expression of the polypeptide(b) expressing the polypeptide in said host cell, and(c) optionally recovering the polypeptide from the nutrient medium orfrom the host cell.

The invention further provides a process to produce a polypeptide by amethod comprising:

(a) transforming an arylsulfatase deficient host cell with an expressionvector, wherein the vector expresses the polypeptide,(b) cultivating the host cell in a nutrient medium, under conditionsconductive to expression of the polypeptide(c) expressing the polypeptide in the host cell, and(d) optionally recovering the polypeptide from the nutrient medium orfrom the host cell.

The invention further provides a process to produce a polypeptide by amethod comprising:

(a) cultivating a host cell in a nutrient medium that prohibits theproduction of arylsulfatase and under conditions conductive toexpression of the polypeptide(b) expressing the polypeptide in said host cell, and(c) optionally recovering the polypeptide from the nutrient medium orfrom the host cell.

The invention further provides a process to produce a polypeptide by amethod comprising:

(a) transforming a host cell with an expression vector, wherein thevector expresses the polypeptide,(b) cultivating the host cell in a nutrient medium that prohibits theproduction of arylsulfatase and under conditions conductive toexpression of the polypeptide(c) expressing the polypeptide in the host cell, and(d) optionally recovering the polypeptide from the nutrient medium orfrom the host cell.

In a preferred embodiment, the polypeptide is an enzyme. In a preferredembodiment, a process for preparing an enzyme preparation is provided,said process comprising preparing an enzyme by a process as disclosedherein, and recovering an enzyme preparation from the nutrient medium orfrom the host cell.

Further disclosure is given below.

Fermentative Repression

In a preferred embodiment, the enzyme preparation may be produced usingan industrial host strain that is cultivated in a growth medium thatlimits or prohibits the production of arylsulfatases. For Pseudomonasaeruginosa it has been described that after cultivation in a mediumcontaining an excess of sulphate as sole sulphur source, no significantlevel of arylsulfatase could be detected, while the use ofethanesulfonate as sole sulphur source leads to the production ofsignificant amounts of arylsulfatase activity (Beil et al. (1995) Eur.J. Biochem. 229, 385-394). Therefore, it is conceivable that using anexcess of sulphate in the fermentation medium also has a repressingeffect on the production of arylsulfatase activity in industrially moreimportant micro-organisms. Growth of the enzyme production organism in amedium containing an excess of sulphate as sulphur source mighttherefore lead to the production of preferable enzyme products with areduced amount of arylsulfatase activity. With an excess of sulphate inthe medium it is meant here that a significant amount of free sulphateis still left in the broth after growth of the micro-organism has beencompleted. It is not required for this invention that sulphate is thesole sulphur source in the growth medium, as long as the molar amount ofsulphate in the growth medium is higher than the molar amount of anyother sulphur containing substance, during the complete growth period.Additionally, also cysteine or thiocyanate might be used instead ofsulphate, as preferred sulphur source in the repression of arylsulfataseactivity. Additionally, it is also relevant to have a significant amountof sulphate, or another repressing sulphur source, in all solutionsduring washing, storage and other down-stream-processing steps, toprevent the derepression of arylsulfatase activity in the broth, evenafter the end of the fermentation.

Classical Strain Improvement

An arylsulfatase deficient strain may be obtained by genetic engineeringusing recombinant genetic manipulation techniques, submitting the hostto mutagenesis, or both. Modification or inactivation of the genescoding for arylsulfatase of the present invention may result fromsubjecting the parent cell to mutagenesis and selecting for mutant cellsin which the ability to express arylsulfatases has been reduced bycomparison to the parental cell. The mutagenesis, which may be specificor random, may be performed, for example, by use of a suitable physicalor chemical mutagenizing agent, by use of a suitable oligonucleotide, orby subjecting the DNA sequence to PCR-generated mutagenesis.Furthermore, the mutagenesis may be performed by use of any combinationof these mutagenizing agents.

Examples of a physical or chemical mutagenizing agent suitable for thepresent purpose include gamma or ultraviolet (UV) radiation,hydroxylamine, N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), O-methylhydroxylamine, nitrous acid, ethyl methane sulphonate (EMS), sodiumbisulphite, formic acid, and nucleotide analogs. When such agents areused, the mutagenesis is typically performed by incubating the parentcell to be mutagenized in the presence of the mutagenizing agent ofchoice under suitable conditions, and selecting for mutant cellsexhibiting reduced expression of the gene. Alternatively, such strainsmay be isolated using genetic techniques such as hybridization ormating, and protoplast fusion or any other classical genetic techniqueto induce genetic diversity. The arylsulfatase deficient strain obtainedmay be subsequently selected by monitoring the expression level of thearylsulfatase. Optionally, the arylsulfatase deficient strain issubsequently selected by measuring the expression level of a given geneof interest to be expressed in the host cell. Selection of strainshaving reduced arylsulfatase activity may be done by directly measuringarylsulfatase activity in culture broth, in culture supernatant, inpermeabilized cells, or in cell lysate. For measuring arylsulfataseactivity it is possible to optionally permeabilize cells of theindustrial production strain, incubate with a fluorescent substrate(such as 4-methylumbelliferone-sulphate (MUS)), until the substrate hasbeen taken up by the cells, and screen for cells with lowerarylsulfatase activity by measuring the decrease in fluorescence. Suchmeasurement may be done directly using a conventional fluorimeter inindividual cultures, or preferably be done by flow cytometry in such away that the cells with low fluorescence can be sorted out and used forfurther cultivation. Cells used in such a procedure may or may not bemutagenized prior to the incubation with fluorescent substrate.

Alternatively, strains having reduced arylsulfatase activity may beisolated by selection for strains that are not able to grow on sulphateesters of alkylesters (such as cresyl sulphate or ethanesulfonate) assole sulphur source in the growth medium.

Isolation of suitable strains according to the invention may requireseveral rounds of classical genetic techniques to be applied, especiallyin industrial production strains that are not haploid, but diploid,aneuploid or have a different ploidy such is the case with manyindustrial yeast strains, or in case the industrial production straincontains multiple genes coding for arylsulfatase, such is the case infungi.

Recombinant DNA Techniques

Alternatively, industrial production strains that have a reduced amountof arylsulfatase activity may be constructed using recombinant DNAtechnology. Several techniques for gene inactivation or gene disruptionare described in the art, such as one-step gene disruption, markerinsertion, site directed mutagenesis, deletion, RNA interference,anti-sense RNA, and others, and may all be used to lower, inhibit ordisturb the synthesis of the arylsulfatase activity in order to obtain aindustrial production strain with decreased arylsulfatase activity. Alsothe inactivation of arylsulfatase by altering the control sequence(s)directing the expression of the arylsulfatase gene are part of thepresent invention. An example thereof is the lowering of the promoteractivity by gene disruption.

Using modern genetic modification techniques, one can obtain arecombinant arylsulfatase deficient strain, preferably by disturbing agene coding for arylsulfatase activity, more preferably by inserting amarker gene into a gene coding for arylsulfatase activity, mostpreferably by removal of part or all of the arylsulfatase coding regionfrom the genome. Methods to perform such gene inactivations have beendescribed for many different mincro-organisms and are known to thoseskilled in the art (see i.e. EP357127) and is also described in Example8. Expression of arylsulfatases in the mutant cell may thereby bereduced or eliminated. Dependent on the host strain that is modifiedusing these techniques, the procedure should be repeated several timesto remove all or most of the arylsullfatase coding sequences.

Modification or inactivation of a host gene such as arylsulfatase may beperformed by established antisense techniques using a nucleotidesequence complementary to the nucleotide sequence of the gene. Morespecifically, expression of the gene may be reduced or eliminated byintroducing a nucleotide sequence complementary to the nucleotidesequence, which may be transcribed in the cell and is capable ofhybridizing to the mRNA produced in the cell. Under conditions allowingthe complementary antisense nucleotide sequence to hybridize to themRNA, the amount of protein translated is thus reduced or eliminated.Examples of expressing an antisense RNA is provided by Ngiam et al.(Appl. Environ. Microbiol. 66:775-782, 2000) and Zrenner et al. (Planta190:247-252, 1993).

Modification, downregulation, or inactivation of a host gene may beobtained via RNA interference (RNAi) techniques (FEMS Microb. Lett.237:317-324, 2004). More specifically, expression of the gene by afilamentous fungal cell may be reduced or eliminated by cloningidentical sense and antisense portions of the nucleotide sequence, whichexpression is to be affected, behind each other with a nucleotide spacerin between, inserting into an expression vector, and introducing theexpression vector into the cell where double-stranded RNA (dsRNA) may betranscribed and then processed to shorter siRNA that is able tohybridize to target mRNA. After dsRNA is transcribed, formation of small(21-23) nucleotide siRNA fragments will lead to a targeted degradationof the mRNA, which is to be affected. The elimination of the specificmRNA can be to various extents. The RNA interference techniquesdescribed in WO 2005/05672 and WO 2005/026356 may be used formodification, downregulation, or inactivation of the host gene.

The arylsulfatase deficient strain, which has been modified orinactivated by any of the methods described above and produces fewerarylsulfatase activity than the parent cell when cultured underidentical conditions as measured using the same assays as definedbefore, may harbor another nucleotide sequence.

Such industrial production strains with decreased arylsulfatase activityisolated or constructed by classical genetic techniques or recombinantDNA technology may be used for relevant industrial processes thatrequire the final product to lack off-flavour. Preferably these strainsare used for the production of industrially relevant enzymes. Morepreferably these strains are used for the production of enzymes that areused in the food industry, even more preferably these enzymes are usedin processing of dairy products. Most preferably such industrialproduction strains with decreased arylsulfatase activity are used forthe production of lactase.

Host Strains

Suitable industrial host strains are preferably prokaryoticmicroorganisms such as bacteria, or more preferably eukaryoticorganisms, for example fungi, such as yeasts or filamentous fungi, orplant cells. Bacteria from the genus Bacillus are very suitable as hostsbecause of their capability to secrete proteins into the culture medium.Other bacteria suitable as hosts are those from the genera Streptomycesand Pseudomonas. A preferred yeast host cell for the expression of a DNAsequence encoding the enzyme of interest is one of the genusSaccharomyces, Kluyveromyces, Hansenula, Pichia, Yarrowia, orSchizosaccharomyces. More preferably, a yeast host cell is selected fromthe group consisting of the species Saccharomyces cerevisiae,Kluyveromyces lactis (also known as Kluyveromyces marxianus var.lactis), Hansenula polymorpha, Pichia pastoris, Yarrowia lipolytica, andSchizosaccharomyces pombe.

Most preferred for the expression of an enzyme are, however, filamentousfungal host cells. Preferred filamentous fungal host cells are selectedfrom the group consisting of the genera Aspergillus, Trichoderma,Fusarium, Disporotrichum, Penicillium, Acremonium, Neurospora,Thermoascus, Myceliophtora, Sporotrichum, Thielavia, and Talaromyces.More preferably a filamentous fungal host cell is of the speciesAspergillus oyzae, Aspergillus sojae or Aspergillus nidulans or is of aspecies from the Aspergillus niger Group (as defined by Raper andFennell, The Genus Aspergillus, The Williams & Wilkins Company,Baltimore, pp 293-344, 1965). These include but are not limited toAspergillus niger, Aspergillus awamori, Aspergillus tubigensis,Aspergillus aculeatus, Aspergillus foetidus, Aspergillus nidulans,Aspergillus japonicus, Aspergillus oryzae and Aspergillus ficuum, andalso those of the species Trichoderma reesei, Fusarium graminearum,Penicillium chrysogenum, Acremonium alabamense, Neurospora crassa,Myceliophtora thermophilum, Sporotrichum cellulophilum, Disporotrichumdimorphosporum and Thielavia terrestris.

Examples of preferred industrial production strains within the scope ofthe present invention are fungi such as Aspergillus species (inparticular those described in EP-A-184,438 and EP-A-284,603) andTrichoderma species; bacteria such as Bacillus species (in particularthose described in EP-A-134,048 and EP-A-253,455), especially Bacillussubtilis, Bacillus licheniformis, Bacillus amyloliquefaciens,Pseudomonas species; and yeasts such as Kluyveromyces species (inparticular those described in EP-A-096,430 such as Kluyveromyces lactisand in EP-A-301,670) Saccharomyces species, such as Saccharomycescerevisiae, or Pichia pastoris, Hansenula polymorpha, Candida utilis orYarrowia lipolytica. The current invention most preferably relates tothe production of lactase lacking arylsulfatase activity byKluyveromyces lactis.

Arylsulfatase deficient strains suitable for the production of a givenpolypeptide or enzyme in an industrial setting have been isolated,wherein surprisingly the arylsulfatase deficient strain produce at leastthe same amount of polypeptide or enzyme as the wild type strain theyoriginate from under the same culture conditions.

Preferably, the arylsulfatase deficient strains of the invention arestrains have less than 50% of the detectable intracellular orextracellular arylsulfatase activity as detected in a model reaction(see experimental information in the Example 2). More preferably, thearylsulfatase deficient strains of the invention are strains having lessthan 50% of the intracellular arylsulfatase activity. More preferably,the arylsulfatase deficient strains of the invention are strains havingan intracellular arylsulfatase activity, which is less than 25% of theintracellular arylsulfatase activity of the wild type strain theyoriginate from as detected in a model reaction, preferably less than10%, more preferably less than 5%, more preferably less than 1% and mostpreferably the arylsulfatase activity is undetectable in thearylsulfatase deficient strains.

In this application, K. lactis strain CBS 2359 is taken as a referenceof wild type arylsulfatase levels obtainable in an K. lactis culture, asa reference of wild type polypeptide level obtainable in an K. lactisculture and as a reference of intracellular arylsulfatase activityobtainable in an K. lactis culture. Arylsulfatase deficient K. lactisstrains are defined as strains that produce less arylsulfatase activitythan the K. lactis strain CBS 2359 under the same culture conditions.Preferably, the arylsulfatase deficient strain is a K. lactis strainshaving less than 50% of the intracellular arylsulfatase activity of theK. lactis CBS 2359 strain as detected in a model reaction. Morepreferably, the arylsulfatase deficient K. lactis strains of theinvention are strains having an intracellular arylsulfatase activity,which is less than 25% of the intracellular arylsulfatase activity ofthe K. lactis CBS 2359 strain they originate from as detected in a modelreaction, preferably less than 10%, more preferably less than 5%, morepreferably less than 1% and most preferably the arylsulfatase activityis undetectable in the arylsulfatase deficient K. lactis strains.According to a preferred embodiment of the invention, the arylsulfatasedeficient K. lactis strain used has been obtained by applying the methoddefined later in this application.

A large variety of systems for detection of polypeptide are known to theskilled person. Detection systems include any possible assay fordetection of polypeptide or enzymatic activity. By way of example theseassay systems include but are not limited to assays based oncolorimetric, photometric, fluorometric, turbidimetric, viscosimetric,immunological, biological, chromatographic, and other available assays.

Preferably, if the polypeptide produced is an enzyme, the amount ofactive enzyme produced is determined by measurement of its activity in amodel reaction (see example 2).

According to a further preferred embodiment, the arylsulfatase deficientstrain of the invention is characterized by the fact that when thisstrain has been transformed with an expression construct comprising agene coding for a polypeptide, said strain produces at least the amountof the polypeptide the wild type strain it originates from would produceunder the same culture conditions, when the wild type strain has alsobeen transformed with the same expression construct as the arylsulfatasedeficient strain. Preferably, the arylsulfatase deficient strains of theinvention are strains that produce the same amount or more of a givenpolypeptide than the wild type strain they originate from under the sameculture conditions. More preferably, the arylsulfatase deficient strainproduces more of a given polypeptide than the wild type strain theyoriginate from under the same culture conditions.

Production of Other Native or Heterologous Polypeptides and OtherSequences

According to yet another embodiment, the present invention relates tomethods of transcribing a nucleotide sequence in a host cell, whereinthe transcribed sequence encodes a desired polypeptide or is afunctional nucleic acid molecule, comprising:

(a) cultivating, in a nutrient medium, a host cell comprising (i) apromoter, (iv) a downstream nucleotide sequence which encodes apolypeptide, (iii) a translational stop signal and (iv) atranscriptional stop signal,(b) expressing the polypeptide in the host cell, and(c) optionally, recovering the polypeptide from the nutrient medium orfrom the host cell.

The polypeptide produced may be sensitive to protease degradation. Inthis case, a mutant host cell which is protease deficient will be used.The arylsulfatase deficient strain is preferably produced according tothe method of the present invention. The arylsulfatase deficient strainmay be grown or maintained in a nutrient medium suitable for productionof the desired polypeptide using methods known in the art. For example,cells may be plated on a solid substrate, shaken in a flask, cultivatedin small-scale or large-scale fermentation (including continuous, batch,fedbatch, or solid-state fermentation) in laboratory or industrialfermentors in a suitable medium and under conditions allowing thepolypeptide to be expressed and/or isolated. Cultivation takes place ina suitable nutrient medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art (see, e.g., Bennett &LaSure, eds., More Gene Manipulations in Fungi, Academic Press, CA,1991). Suitable media are available from commercial suppliers or may beprepared using published compositions (e.g., in catalogues of theAmerican Type Culture Collection). If the polypeptide is secreted intothe nutrient medium, the polypeptide can be recovered directly from themedium. If the polypeptide is not secreted, it can be recovered fromcell lysates.

The resulting polypeptide may be isolated by methods known in the art.For example, the polypeptide may be isolated from the nutrient medium byconventional procedures including, but not limited to, centrifugation,filtration, extraction, spray drying, evaporation, or precipitation. Theisolated polypeptide may then be further purified by a variety ofprocedures known in the art including, but not limited to,chromatography (e.g., ion exchange, affinity, hydrophobic,chromatofocusing, or size exclusion), electrophoresis (e.g., preparativeisoelectric focusing), differential solubility (e.g., acetone orammonium sulfate precipitation), or extraction (e.g., chaotrope, salt,or pH). See, e.g., Janson & Ryden, eds., Protein Purification, VCHPublishers, New York, 1989.

The polypeptide may be detected using methods known in the art that arespecific for the polypeptide. These detection methods may include use ofspecific antibodies, formation of an enzyme product, disappearance of anenzyme substrate, or SDS-PAGE. For example, an enzyme assay may be usedto determine the activity of the polypeptide. Procedures for determiningenzyme activity are known in the art for many enzymes.

The polypeptide may be any polypeptide whether native or heterologous tothe arylsulfatase deficient strain. The term “heterologous polypeptide”is defined herein as a polypeptide, which is not produced by a wild-typestrain. The term “polypeptide” is not meant herein to refer to aspecific length of the encoded produce and therefore encompassespeptides, oligopeptides and proteins. The nucleotide sequence encoding aheterologous polypeptide may be obtained from any prokaryote, eukaryote,or other source and may be a synthetic gene. The term “obtained from” asused herein in connection with a given source shall mean that thepolypeptide is produced by the source or by a cell in which a gene fromthe source has been inserted.

The desired polypeptide may be an antibody or antigen-binding portionthereof, antigen, clotting factor, enzyme, peptide hormone or variantthereof, receptor or ligand-binding portion thereof, regulatory protein,structural protein, reporter, transport protein, intracellular protein,protein involved in a secretory process, protein involved in a foldingprocess, chaperone, peptide amino acid transporter, glycosylationfactor, or transcription factor. The polypeptide may be secretedextracellularly into culture medium.

There is no limitation to a specific enzyme. Preferred enzymes aredisclosed in the remainder of the specification and examples.

Alternatively the polypeptide may be an intracellular protein or enzymesuch as, for example, a chaperone, protease, or transcription factor. Anexample of this is described by Punt et al. (Appl. Microbiol.Biotechnol. 50:447-454, 1998). This can be used for example to improvethe efficiency of a host cell as protein producer if this polypeptide,such as a chaperone, protease, or transcription factor, is known to be alimiting factor in protein production.

In the methods of the present invention, the arylsulfatase deficientstrain may also be used for the recombinant production of polypeptides,which are native to the cell. The native polypeptides may berecombinantly produced by, e.g., placing a gene encoding the polypeptideunder the control of a different promoter to enhance expression of thepolypeptide, to expedite export of a native polypeptide of interestoutside the cell by use of a signal sequence, and to increase the copynumber of a gene encoding the polypeptide normally produced by the cell.The present invention also encompasses, within the scope of the term“heterologous polypeptide”, such recombinant production of polypeptidesnative to the cell, to the extent that such expression involves the useof genetic elements not endogenous to the cell, or use of endogenoussequence elements which have been manipulated to function in a mannerthat do not normally occur in the filamentous fungal cell. Thetechniques used to isolate or clone a nucleotide sequence encoding aheterologous polypeptide are known in the art and include isolation fromgenomic DNA, preparation from cDNA, or a combination thereof.

In the methods of the present invention, heterologous polypeptides mayalso include a fused or hybrid polypeptide in which another polypeptideis fused at the N-terminus or the C-terminus of the polypeptide orfragment thereof. A fused polypeptide is produced by fusing a nucleotidesequence (or a portion thereof) encoding one polypeptide to a nucleotidesequence (or a portion thereof) encoding another polypeptide.

Techniques for producing fusion polypeptides are known in the art, andinclude, ligating the coding sequences encoding the polypeptides so thatthey are in frame and expression of the fused polypeptide is undercontrol of the same promoter (s) and terminator. The hybrid polypeptidesmay comprise a combination of partial or complete polypeptide sequencesobtained from at least two different polypeptides wherein one or moremay be heterologous to the mutant fungal cell. An isolated nucleotidesequence encoding a heterologous polypeptide of interest may bemanipulated in a variety of ways to provide for expression of thepolypeptide. Expression will be understood to include any step involvedin the production of the polypeptide including, but not limited to,transcription, posttranscriptional modification, translation,posttranslational modification, and secretion. Manipulation of thenucleotide sequence encoding a polypeptide prior to its insertion into avector may be desirable or necessary depending on the expression vector.The techniques for modifying nucleotide sequences utilizing cloningmethods are well known in the art.

The DNA sequence encoding the polypeptide to be produced may be operablylinked to appropriate DNA regulatory regions to ensure a high level ofexpression of said DNA sequence and preferably a high secretion level ofsaid polypeptide. If the polypeptide to be produced is native to thearylsulfatase deficient strain, its native secretion signal ispreferably used. Alternatively, if the polypeptide to be produced is notnative to the arylsulfatase deficient strain, a fusion construct ispreferably made comprising i.e. the glucoamylase gene of Aspergillusniger fused to the heterologous gene to be produced. According to apreferred embodiment of the invention, the regulatory regions of theAspergillus oryzae alpha amylase gene are used. According to a morepreferred embodiment of the invention, the regulatory regions of the A.niger glucoamylase gene are used. According to a more preferredembodiment of the invention, the regulatory regions of the K. lactislactase gene are used. The DNA construct may also comprise a selectablemarker. Alternatively, the selectable marker may be present on a secondDNA construct. By way of example these markers include but are notlimited to amdS (acetamidase genes), auxotrophic marker genes such asargB, trpC, or pyrG and antibiotic resistance genes providing resistanceagainst e.g. phleomycin, hygromycin B or G418. Preferably, the markergene is the acetamidase gene from Aspergillus nidulans. More preferably,the acetamidase gene from Aspergillus nidulans is fused to the gpdApromoter. More preferably, the acetamidase gene from Aspergillusnidulans is fused to the Saccharomyces cerevisiae ADH1 promoter.

A method was developed for obtaining arylsulfatase deficient strainwhich are suitable for producing high yields of a polypeptide and whichcan be used as polypeptide producers in an industrial setting. Thepolypeptide may be homologous or heterologous for said arylsulfatasedeficient strain. In case of a heterologous polypeptide or enzyme, thewild type strain on which the method of the invention is applied mayhave been earlier transformed to express a gene coding for suchpolypeptide or enzyme as has been described earlier in the description.Such arylsulfatase deficient strains produce at least the amount ofpolypeptide the wild type strains they originate from produce under thesame culture conditions. Alternatively, the construction of thearylsulfatase deficient strain can be performed prior to thetransformation with a gene coding for such polypeptide or enzyme as hasbeen described earlier in the description.

According to an embodiment of the invention, polypeptides areconsequently produced in a host cell of the present invention with areduced arylsulfatase phenotype, which cell is a mutant of a parent celluseful for the production of enzymes useful in the food industry, inwhich the parent cell comprises one or more nucleotide sequencesencoding arylsulfatases and the mutant cell produces less arylsulfataseactivity than the parent cell when cultured under the same conditions.

Preferred features disclosed for one aspect of the invention are alsoapplicable to other aspects of the invention.

The invention will now be elucidated with reference to the followingexamples without however being limited thereto.

LEGEND TO THE FIGURES

FIG. 1: Cloning of the 5′-flank of the K. lactis arylsulfatase gene inTOPO vector

FIG. 2: Cloning of the 3′-flank of the K. lactis arylsulfatase gene inTOPO vector

FIG. 3: Cloning of the 3′-flank of the K. lactis arylsulfatase gene,lacking the SacII site, in TOPO vector

FIG. 4: Combining the 5′-flank and the amdS selection cassette in oneplasmid

FIG. 5: Combining the 5′-flank, 3′-flank and the amdS selection cassettein one plasmid

FIG. 6: Final construction of the arylsulfatase knockout construct

FIG. 7: shows the endoprotease profile using Dabcyl-Edans as substrate.

MATERIALS & METHODS

Activity assay arylsulfatase: Arylsulfatase activity was determinedusing p-nitrophenylsulfate (obtained from Sigma) as a substrate. Foractivity measurements, 0.5 ml of substrate solution (20 mMp-nitrophenylsulfate in 100 mM NaP_(i) buffer pH6.5) was mixed with 0.5ml sample solution containing the arylsulfatase activity. The solutionwas incubate at 37° C. for 3 hours. Than the reaction was stopped byaddition of 1.5 ml 0.5M NaOH. The OD at 410 nm was determined (1 cmpathlength) against a blank experiment in which water was added insteadof sample solution. As reference, a solution was prepared in which theenzyme was added after the reaction was stopped with NaOH. The OD₄₁₀ ofthis reference solution was subtracted from the OD₄₁₀ determined for thesolution in which the enzyme was active for three hours. An arylsulfatase unit (ASU) is expressed as the change in OD₄₁₀*10E6/hr. Forliquid products, the aryl sulfatase activity can expressed as the changein OD₄₁₀*10E6/hr per ml of product. For solid products, the arylsulfatase activity can expresses as the change in OD₄₁₀*10E6/hr per g ofproduct. When the activity of the enzyme of interest is known, thearylsulfatase activity can also be expressed as the as the change inOD₄₁₀*10E6/hr per unit of activity of enzyme of interest.

Activity assay acid lactase: Acid lactase is incubated during 15 minuteswith o-nitrophenyl-beta-D-galactopyranoside (Fluka 73660) at pH 4.5 and37 degrees C. to generate o-nitrophenol. The incubation is stopped byadding 10% sodium carbonate. The extinction of the o-nitrophenolgenerated is measured at a wave length of 420 nm and quantifies acidlactase activity. One acid lactase unit (ALU) is the amount of enzymethat under the test conditions generates 1 micromol of o-nitrophenol perminute.

Activity assay proline-specific endoproteases: Overproduction andchromatographic purification of the proline specific endoprotease fromAspergillus niger was accomplished as described in WO 02/45524. The A.niger proline specific endoprotease activity was tested usingCBZ-Gly-Pro-pNA (Bachem, Bubendorf, Switzerland) as a substrate at 37°C. in a citrate/disodium phosphate buffer pH 4.6. The reaction productswere monitored spectrophotometrically at 405 nM. The increase inabsorbance at 405 nm in time is a measure for enzyme activity.

The activity of proline-specific endoproteases with near neutral pHoptima is established under exactly the same conditions but in this casethe enzyme reaction is carried out at pH 7.0.

The activity of proline-specific dipeptidyl peptidases such as DPP IV isestablished under conditions specified for proline-specificendoproteases with near neutral pH optima, but in this case Gly-Pro-pNAis used as the substrate.

A Proline Protease Unit (PPU) is defined as the quantity of enzyme thatreleases 1 μmol of p-nitroanilide per minute under the conditionsspecified and at a substrate concentration of 0.37 mM.

Activity assay carboxypeptidases: The activity of the A. niger derivedcarboxypeptidase PepG (“CPG”; Dal Degan et al., Appl. Env. Microbiol. 58(1992)2144-2152) was established using the synthetic substrateFA-Phe-Ala (Bachem, Bubendorf, Switzerland) as a substrate. Enzymatichydrolysis of this substrate (1.5 mM FA-Phe-Ala at pH 4.5 and 37 degreesC.) results in a decrease of absorbance which is monitored at awavelength of 340 nm. One unit (CPGU) is the amount of enzyme needed todecrease the optical density at 340 nm by one absorbency unit per minuteunder the test conditions.

Activity Assay Amino Peptidases.

The activity of aminopeptidases is established using the syntheticsubstrate X-pNA in which pNA represents p-nitroanilide and “X” an aminoacid residue. Because different aminopeptidases can have differentselectivities, the nature of amino acid residue ‘X” depends on thecleavage preference of the aminopeptidase activity tested. Thus, “X”represents the residue for which the specific aminopeptidase has thehighest preference. Because many aminopeptidases show the highestreactivity towards Phe, Phe-pNA represents a preferred substrate.Various X-pNA substrates can be obtained from Bachem (Bubendorf,Switzerland). Enzymatic hydrolysis of this substrate (1.5 mM at pH 6.5and 37 degrees C.) results in a color development which is monitored ata wavelength of 410 nm. One unit (APU) is the amount of enzyme needed toincrease the optical density at 410 nm by one absorbency units perminute under the test conditions.

Activity assay esterases/lipases: Esterases and lipases catalyse therelease of free fatty acids from triglycerols. In the present assayglycerol tributyrate is used as the substrate. To establish theesterase/lipase activity, the butyric acid released from tributyrate istitrated with sodium hydroxide to a constant pH of 7.5. Therefore, theamount of sodium hydroxide dosed per time unit in order to keep the pHconstant, is directly proportional to the esterase activity of theenzyme sample

The measurement is carried out using a Radiometer pH-stat unit and thefollowing reagents.

Arabic Gum solution:Consecutively dissolve, while gently stirring, 100 gArabic gum (Sigma) and 500 mg Thymol (ICN) in approximately 800 mLdemineralised water in a 1 L volumetric flask. Make up to one litre withwater and mix. Centrifuge the solution for 15 minutes at 4000 rpm. Theresulting arabic gum solution may be kept in the refrigerator for 2months but should be prepared at least one day before use.

Sodium hydroxide 0.02 mol/l: quantitatively transfer the contents of anampoule containing 0.01 mol/L NaOH into a 500 mL volumetric flask withwater. Make up to volume with water and mix.

SDS/BSA solution: Dissolve, while gently stirring, 1 g SDS (Merck) and 1g BSA (fraction V, Sigma) in approximately 40 mL water. Prevent theformation of foam. Make up the volume to 1 litre with water aftercomplete dissolving of the SDS and BSA. Only use a freshly preparedsolution.

Substrate emulsion: Weigh 50 g glycerol tributyrate in a 600 mL glassbeaker and add 300 mL Arabic gum solution. Prepare an emulsion bystirring 5 minutes at maximum speed with the Ultra Turrax. Adjust the pHto 7.5 with NaOH 0.5 mol/L.

To test esterase/lipase activity of a particular enzyme sample, weigh inapproximately 1 g of enzyme sample. and dissolve in SDS/BSA solution.This sample solution should have a final enzyme content equivalent toapprox. 0.2 to 0.8 NBGE/ml (see further). Keep the sample solution onice until the start of the measurement.

Carry out the measurement by subsequently transferring the followingsolutions into the heated reaction vessels: 20 mL substrate emulsion,5.0 mL water (pre-heated at 40° C.) and allow to pre-heat for 15minutes, then start the measurement by adding 5.0 mL of control sampleor the sample solution and start the VIT 90 esterase program of theRadiometer pH-stat unit.

The esterase/lipase unit (NBGE) is defined as the amount of enzyme thatreleases 1 μmol free fatty acid from glycerol tributyrate per minute ata temperature of 40° C. and pH 7.5 in the following procedure.

EXAMPLES Example 1 Identification of Off-Flavour Compounds in UHT-Milk

Maxilact LG5000 (DSM, Netherlands) was added under sterile conditions tosemi-skimmed UHT milk (Friesche Vlag, Netherlands) to levels of 10,000and 40,000 NLU per liter and incubated for 4 days at room temperature.In the reference experiment, no Maxilact was added. Prior to assessmentof the samples by a taste-panel, a fresh lactase-hydrolyzed milk samplewas prepared by adding 40,000 NLU per litre semi-skimmed milk andincubate for 18 hours at room temperature. Sample analysis was performedat NIZO Food Research (The Netherlands) using the SOIR procedure whichis a common procedure at NIZO Food Research and which includes a sensoryand chemical analysis. Sensory analysis was performed directly on theprepared samples and aliquots of each milk sample were frozen at −25° C.in small portions for further chemical analysis.

Sensory analysis was performed by a 9-membered trained panel. Thereference sample was described as cooked, the other samples wereclassified as not standard UHT milks. The main attributes that describedthe off-flavour were chemical, medicinal, urine/unclean andstable/manure.

Volatile compounds were isolated with a simultaneous high vacuumdistillation near room temperature, creating a watery extract of thesample. The volatile compounds were subsequently isolated from thewatery extract using a dynamic headspace and collected at an absorbant.The isolated compounds were injected into a Gas Chromatograph making useof a thermal desorption and separated on a GC-colomn. The GC-effluentwas evaluated by two trained assessors (GC-sniff) and described in odourterms (olfactometry). Duplicated high and low concentrated GC-Sniffanalyses were carried out by using two different purge times (30 minutesand 24 hours) during dynamic head space sampling. Subsequently the peaks(compounds) indicated during the olfactometric analysis as correspondingwith the off-flavour characteristics of the lactase-treated UHT-sampleswere identified by mass spectrometry. The compounds of interest that mayexplain the cause of the off-flavour were identified as 1) esters (ethylbutanoate); 2) sulphur compounds (dimethyl sulfide, dimethyl trisulfideand benzothiazole); 3) sulfur esters (methyl thioacetate,methylthiobutyrate); 4) 1-octen-3-ol; 5) 2-nonenal; 6) β-damascenone; 7)borneol and 8) p-cresol. The p-cresol could originate from conjugates inmilk. The only compound that was associated with the most offensivesensory attribute ‘medicinal’ was p-cresol. The concentration ofp-cresol in the samples was determined using GC-analysis by addition ofstandard quantities of p-cresol to the samples. The concentration ofp-cresol in the UHT-milk sample 4 days incubation) was estimated at 12μg per litre. This is clearly above the flavour threshold of 1 ppb and 2ppb for air and water respectively (Ha et al, (1991) J Dairy Sci 74,3267-3274). It also is in the range of p-cresol-concentrations commonlyfound in cows milk. The results were confirmed by recombinationexperiments in milk, confirming that p-cresol is responsible for themedicinal off-flavour in lactase-treated UHT-milk.

Example 2 Determination of Aryl-Sulfatase and β-Qalactosidase Activity

Arylsulfatase activity was determined using p-nitrophenylsulfate(obtained from Sigma) as a substrate. For activity measurements, 0.5 mlof substrate solution (20 mM p-nitrophenylsulfate in 100 mM NaP_(i)buffer pH6.5) was mixed with 0.5 ml sample solution containing thearylsulfatase activity. The solution was incubated at 37° C. for 3hours. Than the reaction was stopped by addition of 1.5 ml 0.5M NaOH.The OD at 410 nm was determined (1 cm pathlength) against a blankexperiment in which water was added instead of sample solution. Asreference, a solution was prepared in which the enzyme was added afterthe reaction was stopped with NaOH. The OD₄₁₀ of this reference solutionwas subtracted from the OD₄₁₀ determined for the solution in which theenzyme was active for three hours. The sulfatase activity is expressedas the change in OD₄₁₀*10E6/hr and per NLU. The lactase activity (NLU)for the sample solution was determined as given below.

Lactase activity was determined as Neutral Lactase Units (NLU) usingo-nitrophenyl-β-D-galactopyranoside (ONPG) as the substrate, accordingto the procedure described in FCC (fourth ed, July 1996, p 801-802:Lactase (neutral) β-galactosidase activity).

Example 3 Addition of Aryl-Sulfatase to UHT-Milk

The off-flavour test in milk was performed with commercially availablearylsulfatase (Sigma, Aerobacter aerogenes, type VI; 4.9 mg protein/ml;3.9 arylsulfatase units as defined by Sigma/mg protein). In theexperiment, 50 ml of UHT milk (Campina, The Netherlands) was incubatedwith 1 ml enzyme solution at 30° C. The development of off-flavour wasfollowed by sniffing the sample. The typical off-flavour smell that wasalso described in example 1 for the UHT-milk incubated with lactase wasclearly noticeable after 2 hours of incubation. The smell was moreintense after 17 hours of incubation. Apparently, the aryl-sulfatasegenerated a similar off-flavour as lactase. Based on the findings,described in example 1, this can be explained by the release of p-cresolfrom the conjugate p-cresylsulphate in milk. Experiments were alsoperformed in which acid phosphatase (wheat germ, Sigma, 6 phosphataseunits as defined by Sigma in 40 ml milk) or glucuronidase (from E. coli,Sigma, 6350 glucuronidase units as defined by Sigma per 40 ml milk) wereadded instead of arylsulfatase. In these incubations the typicaloff-flavour did not develop. This suggests that the sulphate conjugatesare the most important conjugates for the formation of off-flavour incows milk, This is consistent with literature findings (Lopez et al(1993) J Agric Food Chem. 41, 446-454). The results do not completelyexclude the presence of other off-flavour compounds, which could begenerated by glucuronidase or acid phosphatase but apparently thesecompounds do not reach levels that are higher than the flavourthresholds.

Example 4 Off-Flavor Test UHT-Milk: Procedure

Semi skimmed UHT milk (Campina, The Netherlands) was incubated with20,000 NLU/L milk during 48 hours at 30° C. The lactase was added via asterile filter under sterile conditions to prevent bacterial infection.The milk was tasted after 48 hours by a trained taste panel and comparedwith a milk solution that was incubated under identical conditions butwithout addition of lactase. A reference solution was prepared brieflybefore tasting by adding 5000 NLU/L milk and incubation for 2 hours at30° C. This sweet milk was used as the reference solution by the tastepanel. The off-flavour is scored by the panel as follows: the blank milkwas set as ‘−’. Low off-flavour products containing a noticeable butlight off-flavour are given ‘+’, whereas products containing higheramounts of off-flavour are expressed as ‘++’ or ‘+++’. The indication‘+++’ indicates a high level of off-flavour, perceived as veryunpleasant. Terms used to characterize the off-flavour were the same asthose described in example 1.

Example 5 Purification of K Lactis Lactase: Removal of Aryl-SulfataseActivity

Maxilact LX5000 (DSM, Netherlands), a commercially available K. lactislactase, was diluted 10 times with water and applied to a Q-Sepharosecolumn (Amersham Biosciences), equilibrated in 55 mM KP_(i) (pH7.0).Loading was continued until lactase activity was detected in therun-through of the column. The column was subsequently washed with 4column volumes of 55 mM KP_(i) (pH7.0), followed by elution of lactasewith 65 mM KP_(i) (pH7.0) containing 0.16M NaCl. Fractions werecollected and assayed for lactase activity. The lactase containingfractions were pooled, and loaded on a butyl Sepharose column (AmershamBiosciences) equilibrated in 55 mM KP_(i) (pH7.) containing 1 M NaSO₄.The lactase was applied to the column in presence of 1M NaSO₄ (pH7.0)until lactase was detected in the run-through of the column. The columnwas washed with 4 column volumes of 55 mM KP_(i) (pH7.) containing 1 MNaSO₄ Lactase was eluted using a 15 column volumes inear gradient from55 mM KP_(i) (pH7.0) containing 1 M NaSO₄ to 55 mM KP_(i) (pH7.0). Theelution profile was monitored by UV-detection (280 nm). Fractions werecollected and assayed for lactase activity. Lactase containing fractionswere pooled, with omission of those fractions that were collected afterthe lactase peak (OD 280 nm) had decreased to 50% of the maximum peakvalue. Omission of these fractions is critical to prevent contaminationof the lactase preparation with aryl-sulfatase. The elution of lactasepartly overlaps with the elution of arylsulfatase. The product wasconcentrated and desalted by ultrafiltration on a 10 kdalton filter andpreserved by addition of glycerol to 50% w/w.

Example 6 Protease Levels in Purified Lactase

Protease activity was determined using a series of substrates with thegeneral formula Glu(EDANS)-Ala-Ala-Xxx-Ala-Ala-Lys(DABCYL). (Xxx: any ofthe 20 natural amino acids). The substrates were obtained from PEPSCAN(Lelystad, The Netherlands), and are internally quenched fluorescentsubstrates. When such peptide substrates are cleaved, this results in afluorescent signal. The appearance of fluorescence therefore signals thepresence of endo-protease activity. Endo-protease activity wasdetermined in 96-wells microtiter plates by adding 50 μl enzyme solutionto 200 μl solution containing 50 ρM of the substrate in 100 mM Tris-Bis(pH 6.7). The reaction mixture was incubated for 10 minutes at 40° C. ina TECAN Genius microtiter plate reader using Magellan4 software.Development of fluorescence was followed in time (excitation filter: 340nm, emission filter: 492 nm). Protease activity was quantified as theslope of the fluorescence line, expressed as RFU/minute/NLU. (RFU:relative fluorescent units, as given by the Genius equipment). NLU-unitsof the enzyme sample are determined as given in example 2. FIG. 7 showsthe enormous reduction in protease activity when LX5000 is purified overthe Q-Sepharose column. The pooled fractions after Q-sepharose (example4) have a factor of at least 5-10 lower protease activity compared tothe starting material (LX5000). Lactase samples in which the RFU/min/NLUis <0.5 for each of the substrates used (see FIG. 1) are defined aspreparations that have low levels of protease activity.

Example 7 Comparison of Non-Purified and Purified Lactase

Several lactase preparations were submitted to the off-flavour testwhich is described in example 4. The lactase preparations differed inaryl-sulfatase content. For each preparation, at least two samples wereused; individual samples varied in aryl-sulfatase activity, and activityranges are indicated in the right column of table 1. The results of theoff-flavour test are given in table 1. Clearly, levels of arylsulfataseactivity are correlated with off-flavour formation. Low levels ofaryl-sulfatase (19 or less, see table 1) do not cause off-flavourformation whereas increasing levels lead to increased off-flavourformation. It is also clear that the lactase preparation afterQ-Sepharose still shows off-flavour development, even though theprotease levels are low (see example 5).

TABLE 1 Off flavour development for various lactase preparations. Arylsulfatase activity Level of off- in preparation flavour delta OD *10E6/hr Lactase preparation formation per NLU Milk without addition −  0Lactase after Q-Sepharose + to ++ 100-300 (pooled fractions; example 4)Lactase GODO YNL-2 +  40-120 (GODO, Japan) Lactase after butyl −  <8-19²sepharose (pooled fractions; example 4) Lactase containing high aryl-+++ 723 sulfatase¹ ¹Fraction with high aryl-sulfatase activity, selectedfrom the Q-Sepharose elution fractions described in example 4. ²thelevel of 8 arylsulfatase units (as defined in example 2) is thedetection limit of the assay. <8 means no arylsulfatase activity wasobserved.

Example 8 Different Commercial Enzyme Preparations Contain ArylsulfataseActivity

Various enzyme products produced from different sources and recovered bydifferent processing routes were collected and were analysed forarylsulfatase activity using the assay specified in the Materials &Methods section. From the results obtained (see Table 2), it is clearthat enzyme preparations obtained from various microorganisms such asAspergillus oryzae, Kluyveromyces lactis, Rhizomucor miehei, Talaromycesemersonii and Trichoderma harzianum can be seriously contaminated witharylsulfatase activity. These enzyme preparations can advantageously bepurified by the process according to the invention.

TABLE 2 Aryl sulfatase activity in various commercial enzymepreparations arylsulfatase activity (in delta OD * 10E6/hr per g or mlProd. of enzyme Enzyme product Supplier batch code organism preparation)Sumizyme FP Shin- U-ES29 A. oryzae 39300 * 10E3 U/g (microbialproteases) Nihon (chem syst.) Sumizyme LP Shin- S-9906-02 A. oryzae14950 * 10E3 U/g (microbial proteases) Nihon Maxilact LG2000 DSM AE0050K. lactis 283 * 10E3 U/ml Acid lactase Amano LAFD1050508 A. oryzae12550 * 10E3 U/g (20 mg) * 3.3 h reaction Lipase F-AP15 Amano LFB A.oryzae 1230 * 10E3 U/g (lipases) 1251507 Piccantase A DSM F5583 (20 mg)R. miehei 250 * 10E3 U/g (microbial esterase/lipase) Filtrase BR-X β-DSM AF0392 T. emmersonii 513 * 10E3 U/ml glucanase (microbialhemicellulases) Oenozyme Elevage DSM KM616001 T. harzianum 1985 * 10E3U/g β-glucanase (microbial hemicellulases)

Example 9 Chromatographic Removal of Arylsulfatase Activity from theProline-Specific Protease from Aspergillus niger Using Ion ExchangeChromatography

In order to remove the arylsulfatase side activities from theproline-specific endoprotease secreted by A. niger (WO 02/046381), anumber of chromatographic resins were screened. Because the isoelectricpoints of the protease and the main secreted arylsulfatase activitiessecreted by A. niger were found to be approximately 0.5 pH units apart,the identification of a chromatographic separation that allows anacceptable separation of the two activities, even under large scale,industrial conditions, is quite demanding.

Finally, the cation exchanger SP Sepharose 6FF and the hydrophobicinteraction (HIC) resin butyl Sepharose 6FF (Amersham BiosciencesEurope) were selected. for further tests. Both resins were tested inTricorn 5/100 columns (CV=2.2 ml) using an ÄKTA Explorer 100 controlledby UNICORN 3.20 and an ÄKTA Purifier controlled by UNICORN 3.21 incombination with a FRAC-950 fraction collector. After elution allfractions generated were tested for proline-specific endoproteaseactivity and arylsulfatase activity using methods specified in theMaterials and Methods section.

TABLE 3 Conditions under which the SP-Sepharose-6FF chromatography wasconducted: Buffer A 20 mM Citrat, 0.085M NaCl, pH 3.0 Buffer B 20 mMCitrat, 1.0M NaCl, pH 3.0 Start conc. B (%)/Start cond. 0/10.7 (mS/cm)Flow rate (ml/min) 0.48 Sample volume (ml) 0.40 Wash volume (CV) 6.1Flow through and wash fraction 1.0 and 11.0 sizes (ml) Gradient 0-40% Bin 10CV; 100% for 3CV Eluate fraction size (ml) 1.0

After pooling of the fractions showing proline-specific activity towardsthe chromogenic peptide Z-Gly-Pro-pNA (Bachem, Bubendorf, Switzerland),arylsulfatase activities of the crude and chromatographically purifiedenzyme preparations were compared. It turned out that in preparationsshowing exactly the same proline-specific activity (9 PPU/ml), thearylsulfatase activity was lowered from 3800*10E3 units/ml in the crudepreparation to less than 30*10E3 units/ml in the chromatographicallypurified preparation.

Example 10

Chromatographic Removal of Arylsulfatase Activity from theProline-Specific Protease from Aspergillus niger Using HydrophobicInteraction Chromatography

The HIC chromatography was conducted under the following conditions. Adiafiltrate of the A. niger derived proline-specific endoprotease havingan activity of 10 PPU/ml was used as the starting material. Thisdiafiltrate was diluted two times with 20 mM citrate buffer containing 2M Na₂SO₄ (pH 4.2, G=121 mS/cm) and was subsequently sterilized byfiltration (0.2 μm) before loading on the column.

TABLE 4 Conditions under which purification of example 10 is performed.Resin Butyl Sepharose 6 FF Column type XK26 Column volume (ml) 107Buffer A 20 mM citrate + 1M Na₂SO₄ (pH 4.2; G = 94 mS/cm) Buffer B 20 mMcitrate + 0.02M Na₂SO₄ (pH 4.2; G = 6 mS/cm) Flow rate (ml/min) 15 (or170 cm/h) Equilibration 0 or 20% buffer B (94 or 82 mS/cm) Sample volume(ml) 76-77 ml (with 1M Na₂SO₄ as end concentration) Wash 20% buffer B(83 mS/cm) for 24 CV Flow through and 38.5 ml and collection of totalwash volume or wash fraction sizes (ml) total selection of flow throughand wash Elution (step) 100% buffer B for 12 or 15 CV Eluate fractionsize (ml) 10 or 50 ml

As the result of a considerable tailing after loading of the enzyme onthe column, a long washing procedure was required to obtain baselineseparation. Finally, the proline-specific endoprotease could be elutedfrom the column with buffer B. The fractions containing theproline-specific proteolytic activity were pooled. Though diluted, thispurified material showed significantly lowered arylsulfatase activity ifcalculated back to the original proteolytic activity demonstrating thatthe proline-specific endoproteolytic activity and the arylsulfataseactivity were effectively separated using this hydrophobic interactionchromatography protocol. Also here it turned out that in preparationsshowing exactly the same proline-specific activity (9 PPU/ml), thearylsulfatase activity was lowered from 3800*10E3 units/ml in the crudepreparation to less than 30*10E3 units/ml in the chromatographicallypurified preparation.

Example 11

The Purified Proline-Specific Endoprotease from A. niger GeneratesCasein Hydrolysates without Off Odors

To test the performance of the chromatographically purifiedproline-specific endoprotease, two casein hydrolysates were preparedusing a chromatographically purified and a non-chromatographicallypurified proline-specific endoprotease in exactly the same protocols.

To a solution containing 100 g/L of sodium caseinate (Murray Goldbern,New Zealand) and water, subtilisin (Protex̂ L; 25 milliliter/gram proteinwas added and incubated for 4 hours at 60° C. and a pH as is. Theprecipitate formed slowly dissolved while stirring. At the end, aclarified solution was obtained with a minor precipitate. Then pH of thesolution was adjusted to pH 4.5 and the liquid was split into equalvolumes. To one of these volumes, 1 PPU of a crude A. niger prolylendopeptidase per gram of casein hydrolysate was added; to the othervolume 1 PPU of a chromatographically purified A. niger prolylendopeptidase. Incubation was continued for 9 hours at 55° C. followedby a 10 kDa ultrafiltration for both solutions. After a further heatinactivation step (5 seconden 120 degrees C.) and a cooling down period,the taste and the odor of the two liquids were evaluated by a panel of 5people trained in detecting and ranking off flavours and off odors inmilk hydrolysates. The panel was unanimous in their conclusion that thehydrolysate prepared with the crude proline-specific endoprotease had acharacteristic, “barn-like” odour and flavor which was missing in thepreparation prepared with the chromatographically purifiedproline-specific endoprotease.

TABLE 5 overview of results of examples 11 and 13 In substrate (duringapplication) In enzyme preparation AS-activity in AS-activity substrateAS-activity (In Delta (in Delta OD * 10⁶ (In Delta OD * 10⁶/hr per perliter of OD * 10⁶/hr per ml) PPU) substrate) Crude preparation 3.8 *10⁶   422 * 10³ 42 * 10⁶ Barn-like “proline-specific” flavour (9 PPU/ml)Purified 30 * 10³  3.3 * 10³ 330 * 10³  No barn-like preparation flavour“proline-specific” (9 PPU/ml) Crude preparation 31.2 * 10⁶   15.1 * 10³3.2 * 10⁶  off-flavour “carboxy- peptidase” (2060 CPG/ml) Crudepreparation 10 * 10³ 4.8 960 No off- “carboxy- flavour peptidase” (2060CPG/ml)

Example 12 Chromatographic Removal of Arylsulfatase Activity from aCarboxypeptidase from Aspergillus niger

Because the isoelectric points of the carboxypeptidase (i.e.p. 4.5) andthe main secreted arylsulfatases from A. niger (i.e.p.'s of 5.0 and 5.4)are close together, chromatographic separation of the two enzymes provedto be quite difficult. However, the following procedure allowed us toobtain a pure carboxypeptidase, free from arylsulfatase activity. Mostimportantly, the method is relatively simple so that it can be carriedout on an industrial scale.

Again a SP-Sepharose FF resin was used. The chromatography was conductedunder the following conditions:

TABLE 6 conditions under which chromatography of example 12 is performedBuffer A: 20 mM NaCitrate + 40 mM NaCl pH 3.1 ± 0.1; conductivity 5.5 ±0.3 mS/cm Buffer B: 20 mM NaCitrate pH 5 ± 0.2; conductivity 3 mS/cmEquilibration: PH 3.1 ± 0.1; 5 cv cond 5.5 ± 0.3 mS/cm Load 3100 U/mlresin 7 cv Washing Buffer A 10  cv Elution buffer Buffer B 4 cvCollected pepG Buffer B 1.3-1.5 cv Caustic cleaning 1M NaOH 2 cv

After the crude enzyme was applied to the column, the column was washedwith buffer A to remove unbound/slightly bound contaminations. Finally,the carboxypeptidase is eluted with buffer B. The peak containingactivity towards the hydrolysis of the chromogenic peptide FA-Phe-Ala-OH(Bachem, Bubendorf, Switserland) was collected. In carboxypeptidasepreparations containing comparable carboxypeptidase activities (2060CPG/g; see Materials and Methods for the activity determination), thearylsulfatase activity decreases from an initial 31200*10E3 units/ml inthe crude enzyme to 10*10E3 units/ml in the purified preparation.

Example 13 The Purified Carboxypeptidase from A. niger Accelerates theAging of Gouda Cheeses without Generating Off-Flavours

Milk was unstandardised and collected from the NIZO. Gouda cheese wasmanufactured using the NIZO method. Briefly: After starter addition,stir for 15-20 minutes. Then add rennet, stir for 3 minutes and set(approx. 45-50 minutes). Cut coagulum using the gradual increase dial.This will take 10 minutes and have a final speed of 8.5. Turn the bladesand stir for another 10 minutes at speed 11. Drain till 120 L remains invat. Add 36 L (30% of remaining volume) of water at 55° C. to achieve anend temperature in the vat of 35.5-35.7° C. while stirring at speed 16.Stir for 60 minutes at speed 16. Collect the curd and rest for 15minutes. Divide the curd over the moulds (weight) and rest the filledmoulds for 30 minutes. Press for 30 minutes at 0.7 bar (add the cheesecode after first pressing), 30 minutes at 1.2 bar and then another 30minutes at 1.7 bar. Turn the curd after each. After pressing, thepressure is removed and the cheeses are rested in the moulds (overnightin the brining room at 13° C.). The cheeses are removed from the mouldsand entered into the brine for 30 hours and turned twice to ensureuniform brining. Ripening at 13° C., 88% humidity. The methodstandardizes at 1.05 fat to protein ratio (equivalent to about 0.85 fatto casein Following pasteurisation, the milk was pumped into the 200 Lcheese vats. Delvo®-TEC UX21A (1.5 U; DSM Food Specialities, Delft, TheNetherlands) was used as starter culture and Maxiren® 600 (55 IMCU/Lmilk; DSM Food Specialities, Delft, The Netherlands) as rennet. Thecheeses were brined for 26 hours and ripened at 13° C., 88% RH. Purifiedand non-purified PepG was added with the rennet at a level of 200CPGU/liter milk.

Following 6 and 24 weeks of ripening, representative samples of a cheesefrom each cheese vat were graded using an internal panel. These sessionshave taken place in a round-the-place manner which means that thegraders are informed of the trial details and afterwards discuss theirfindings which are then summarised.

TABLE 7 results of example 13: 6 weeks (n = 7) Control Young Goudacheese, no off flavours, little bit acid and buttery odour. PurifiedPepG More mature Gouda cheese, farmhouse type flavours, a strong odourand a fuller flavour. Unpurified Like the purified PepG cheese onlybitter notes in the PepG flavour and after taste.

TABLE 8 results of example 13: 24 weeks (n = 6) Control Mature Goudacheese, bit salty Purified PepG Cheese with an intense flavour, touch ofsweetness Unpurified Very piquant flavoured cheese, farmhouse cheese,PepG not in balance, off flavour

A clear effect was found as a result of the addition of PepG, bothpurified and unpurified. The bitter notes and the disbalances recordedfor the cheeses in which the non-purified was used lead to theconclusion that PepG should be purified.

Examples 14-15

In the examples described hereinbelow, standard molecular cloningtechniques such as isolation and purification of nucleic acids,electrophoresis of nucleic acids, enzymatic modification, cleavageand/or amplification of nucleic acids, transformation of E. coli, etc.,were performed as described in the literature (Sambrook et al. (2000)“Molecular Cloning: a laboratory manual”, third edition, Cold SpringHarbor Laboratories, Cold Spring Harbor, N.Y., and Innis et al. (eds.)(1990) “PCR protocols, a guide to methods and applications” AcademicPress, San Diego).

Example 14 Construction of an Arylsulfatase Knock-Out Strain ofKluyveromyces lactis

Isolation of Kluyveromyces lactis Chromosomal DNA:

A 100 ml YEPD (1% Yeast-extract; 1% Bacto-peptone; 2% glucose) shakeflask was inoculated with a single colony of K. lactis CBS 2359 andcultivated for 24 hours at 30° C. shaking at 280 rpm. The amount ofcells was counted using a counting chamber and an amount of culturecorresponding to 4.1*10⁸ cells was used. Extraction of chromosomal DNAwas performed using the Fast DNA Spin Kit supplied by Q-BlOgene(Cat#6540-600). The yeast protocol was used: one homogenizing step usingthe Fastprep FP120 homogenizer (BIO101 Savant) of 40 seconds at speedsetting 6.0 was used. Subsequently the sample was cooled on ice andsubsequently homogenized again using the same conditions.

The purity and yield of the extracted genomic DNA was determined usingthe Nanodrop ND1000 spectrofotometer. It was found that theconcentration of the extract was 114 nanogram/microliter. The A260/280and A260/230 ratio was found to be respectively 1.57 and 0.77.

PCR Amplification of 5′ and 3′ Arylsulfatase Flanks:

5′ flank arylsulphatase primers: DFS-15289 (5′→3′):TCG CCG CGG TTG TCA ACT ATA TTA ACT ATG DFS-15290 (5′→3′):GAT AGA TCA TAG AGT AAC AAT TGG 3′ flank arylsulphatase:DFS-15291 (5′→3′): GCA ACT GAA GGT GGT ATC AAT TG DFS-15292 (5′→3′):CAC CCG CGG CAC CAG ATA ATG GAG GTA G 3′ flank SacII⁻ arylsulphatase:DFS-15291 (5′→3′): GCA ACT GAA GGT GGT ATC AAT TG DFS-15340 (5′→3′):CGG CAC CAG ATA ATG GAG GT

The arylsulfatase flanks were amplified using Phusion High-Fidelity DNAPolymerase, (Finnzymes, Espoo Finland). The K. lactis CBS 2359 genomicDNA was diluted 100 times with Milli-Q water and 5 μl was used as atemplate in a 50 μl PCR mix, according to suppliers' instructions. AHybaid MBS 0.2G PCR block using the following programs:

PCR Program 5′ flank arylsulfatase: Stage 1 (1 cycle) 98° C. 30 s Stage2 (30 cycles) 98° C. 10 s 60° C. 30 s 72° C. 30 s Stage 3 (1 cycle) 72°C. 10 min 4° C. Hold PCR Program 3′ flank arylsulphatase: Stage 1 (1cycle) 98° C. 30 s Stage 2 (30 cycles) 98° C. 10 s 72° C. 30 s 72° C. 30s Stage 3 (1 cycle) 72° C. 10 min 4° C. Hold PCR Program 3′ flank SacII⁻arylsulphatase: Stage 1 (1 cycle) 98° C. 30 s Stage 2 (30 cycles) 98° C.10 s 65° C. 30 s 72° C. 30 s Stage 3 (1 cycle) 72° C. 10 min 4° C. Hold

Construction of an Arylsulphatase Knock-Out Vector:

The obtained 5′-, 3′- and 3′ SacII arylsulfatase flank PCR fragmentswere cloned into the pCR-Blunt II-TOPO vector using the Zero Blunt TOPOPCR Cloning Kit (Invitrogen; Part. no. 45-0245), according to suppliers'instructions. The TOPO cloning reactions were transformed to One ShotTOP10 Chemically Competent E. coli (Invitrogen; Part. no. 44-0301)according to suppliers' instructions. Correct clones were selected basedon restriction pattern analysis using MunI, SacII, XcmI, and DraI; MunI,SacII, EcoRI and EcoRV; MunI, EcoRI, EcoRV and SacII for respectively,5′ TOPO, 3′ TOPO and 3′ SacII⁻ TOPO.

The amdS cassette was isolated from the pKLAC1 vector (New EnglandBiolabs). The pKLAC1 plasmid was transformed to chemically competentdam-/dcm- E. coli cells (New England Biolabs; Cat. No C2925H) and theun-methylated plasmid was isolated.

Large plasmid DNA batches of 5′ TOPO, 3′ TOPO, 3′ SacII⁻ TOPO and pKLAC1vector were isolated from overnight LBC cultures containing 50 μg/mlKanamycin using the GeneElute Plasmid MidiPrep Kit (Sigma; Cat. No.NA0200).

The pKLAC1 vector was digested SaII and XbaI and the 5′ TOPO vector wasdigested XbaI and XhoI. Digests were purified using the NucleospinExtractII Kit (Machery Nagel) according to suppliers' instructions.

The Sall/XbaI digested amdS cassette was ligated into the XbaI/XhoIdigested 5′ TOPO vector using the Quick ligation Kit (New EnglandBiolabs; Cat. No. M2200S) according to suppliers' instructions. Theligation mix was transformed to One Shot TOP10 Chemically Competent E.coli (Invitrogen; Part. no. 44-0301) according to suppliers'instructions. A correct clone was selected based on restriction patternanalysis using MunI, EcoRI and SacI. This resulted in the followingvector: 5′amdS TOPO vector (FIG. 1)

A large batch of the 5′amdS TOPO plasmid was isolated from overnight LBCcultures containing 50 μg/ml Kanamycin using the GeneElute PlasmidMidiPrep Kit (Sigma; Cat. No. NA0200) according to suppliers'instructions. The 5′amdS TOPO vector was digested MunI and AscI and the3′ SacII⁻ TOPO vector was digested MunI and EcoRI. The MunI/EcoRI 3′SacII⁻ TOPO fragment was isolated and purified by means of gelextraction. An electrophoresis was performed on 1% agarose in TAE buffercontaining SYBR Safe DNA Stain (Invitrogen; Cat. No. S33102), accordingto suppliers' instructions. The fragment was visualized using the darkreader transilluminator (Clare Chemical Research; Cat. No. DR-45M),excised from the gel and extracted from the agarose using the NucleospinExtractII kit (Machery Nagel; Cat. No. 740 609.250) according tosuppliers' gel extraction protocol.

The 5′amdS TOPO vector was digested MunI and AscI and purified using theNucleospin ExtractII Kit (Machery Nagel) according to suppliers' PCRpurification protocol. Subsequently the MunI/ASCI digested 5′amdS TOPOvector was dephosphorylated using Shrimp Alkaline Phosphatase (Roche;Cat. No. 1 758 250) according to suppliers' instructions.

The MunI/EcoRI 3′ SacII⁻ TOPO fragment was ligated into thedephosphorylated MunI/AscI digested 5′ amdS TOPO vector using the Quickligation Kit (New England Biolabs; Cat. No. M2200S) according tosuppliers' instructions. The ligation mix was transformed to chemicallycompetent dam-/dcm- E. coli cells, (New England Biolabs; Cat. No C2925H)according to suppliers' instructions. A correct clone was selected basedon restriction pattern analysis using EcoRI and EcoRV. This resulted inthe following vector: 5′ amdS 3′ SacII-TOPO vector.

A large batch of 5′amdS 3′ SacII⁻ TOPO vector was isolated fromovernight LBC cultures containing 50 μg/ml Kanamycin using the GeneElutePlasmid MidiPrep Kit (Sigma; Cat. No. NA0200) according to suppliers'instructions.

The 5′amdS 3′SacII⁻ vector was digested with XbaI. The 3′ TOPO vectorwas digested with XbaI and SpeI. Digests were purified using theNucleospin ExtractII Kit (Machery Nagel) according to suppliers'instructions. The XbaI/SpeI 3′ TOPO fragment was isolated by means ofgel extraction, as described above. The XbaI digested 5′amdS 3′SacII⁻vector was dephosphorylated using Shrimp Alkaline Phosphatase, Roche(Cat. No. 1 758 250) according to suppliers' instructions. The XbaI/SpeI3′ fragment was ligated in the dephoshorylated XbaI digested 5′ amdS 3′SacII⁻ vector using the Quick ligation Kit (New England Biolabs; Cat.No. M2200S) according to suppliers' instructions. The ligation mix wastransformed to One Shot TOP10 Chemically Competent E. coli (Invitrogen;Part. no. 44-0301). A correct clone was selected based on restrictionpattern analysis using MfeI, KpnI, EcoRI, SacII, ScaI. This resulted inthe final K. lactis arylsulphatase knock-out vector (FIG. 3).

A large batch of the arylsulphatase knock-out vector was isolated fromovernight LBC cultures containing 50 μg/ml Kanamycin using the GeneElutePlasmid MidiPrep Kit (Sigma; Cat. No. NA0200) according to suppliers'instructions. The K. lactis arylsulphatase knock-out vector was digestedwith SacII so the linear knock-out cassette would be obtained, lackingthe TOPO vector part. The digest was purified using the NucleospinExtractII Kit (Machery Nagel) according to suppliers' instructions.

Transformation of K. lactis CBS 2359 with Arylsulphatase Knock-OutVector

A 100 ml YEPD culture of K. lactis CBS2359 was incubated at 30° C.,shaking at 280 rpm for 24 hours. This culture was used to inoculate a100 ml YEPD culture which was grown under the same conditions until anOD610 between 0.5 and 0.8 was reached.

Cells were harvested by means of centrifugation for 5 minutes at 1559 gand 4° C. The cell pellet is washed with 50 ml sterile electroporationbuffer (EB): 10 mM Tris pH 7.5, 9.2% (w/v) Sucrose, 1 mM MgCl₂ at 4° C.The cell pellet was resuspended in 50 ml YEPD containing 25 mM DTT and20 mM HEPES buffer pH 8.0 at room temperature. The cells were incubated30 minutes at 30° c. without shaking. The cells were harvested by meansof centrifugation for 5 minutes at 1559 g and 4° C. and washed with 10ml ice cold EB. The cells were again pelletted by means ofcentrifugation for 5 minutes at 1559 g and 4° C. and resuspended in 0.1ml ice cold EB. The cell suspension was distributed in 40 microliteraliquots in 1.5 ml eppendorf tubes. To one aliquot of cells 0.2-1.0microgram (1-5 microliter) of the linear knock out construct was added,mixed by pipetting and incubated on ice for 15 minutes. The cell-DNA mixwas added to a chilled electroporation cuvette with a 2 mm gap size(BTX; Part. No. 45-0125). Electroporation was performed on a BioRadelectroporator composed of a Gene Pulser (BioRad, Model No. 1652077) anda Pulse Controler (BioRad, Model No. 1652098) using the followingsettings: 1000 V, 400 Ohm and 25 μF. Immediately after electroporation 1ml YEPD was added and the cells were transferred to a sterile 12 ml tubeand incubated during 2 hours in an shaking incubator at 30° C. The cellswere pelletted for 5 minutes at 1559 g and washed in fysiologic salinesolution (0.85% (w/v) sodium chloride). The cells were again pellettedand resuspended in 1 ml fysiologic saline solution. Several aliquots of25 μl, 50 μl and 100 μl were plated on selective amdS agar plates: 1.25%(w/v) agar, 1.17% (w/v) Yeast Carbon Base, 30 mM phosphate buffer pH 6.8and 5 mM acetamide. Plates were incubated for 2 days at 30° C. followedby 2 days incubation at room temperature.

Colonies were selected and purified by streaking them on YEPD agarplates so single colonies would appear and incubated at 30° C. for 24hours. These single colonies were tested for targeted integration of theknockout construct using a colony PCR with oligonucleotides targetedagainst the amdS cassette and downstream of the integrated knock outconstruct. Colony material was suspended in 20 mM NaOH, 0.2% (w/v) andincubated for 5′ at 98° C. The cell suspension was diluted 2 times withwater and 2.5 microliter was used directly as template in a 25microliter PCR reaction using Phusion High-Fidelity DNA Polymerase(Finnzymes; Espoo Finland; Product code F-530S) according to suppliers'instructions.

Fw 3′ amdS: GAC AAT TGA TAC CAC CTT CAG TTGRv downstream: CTG GGA AAT GTG GTG ACT CCA TA

Program Targeting PCR:

Stage 1 (1 cycle) 98° C. 30 s Stage 2 (30 cycles) 98° C. 10 s 68° C. 30s 72° C. 30 s Stage 3 (1 cycle) 72° C. 10 min 4° C. Hold

PCR was analysed on 1% agarose gels and targeted transformants that showa clear amplified band were selected. The arylsulfatase knockout strainswere named 2359ΔARY1-10, and stored until further use.

Example 15 Detection of Arylsulfatase Activity in 2359ΔARY

Motherstrain CBS 2359 and strain 2359ΔARY were all cultivated inshakeflask in 100 ml YEP+2% galactose for 3 days at 30° C. Biomass wascollected by centrifugation for 5 minutes at 1559 g and 4° C. Biomasswas washed twice with ice-cold water to remove medium components. Yeastbiomass was treated with Yeast Protein Extraction reagent (Y-PER)according the instructions of the manufacturer (Pierce), to extractintracellular enzymes like arylsulfatase. It will be clear to thoseskilled in the art that other yeast lysis protocols can be used toextract arylsulfatase activity, like mechanical sheering with glasbeads,or enzymatic treatment to dissolve the cell wall with e.g. Zymolyase(see i.e. Glover and Hames, DNA cloning 2—a practical approach, IRLPress 1995).

Arylsulfatase was measured in the extract using the method described inExample 2. From this experiment it became clear that while themotherstrain contained an appreciable amount of arylsulfatase activity,no such activity could be detected in the 2359ΔARY strain. When theβ-galactosidase (lactase) activity was measured in this extractaccording to Example 2, no difference in lactase activity could bedetected between the wild type strain CBS 2359 and the mutant strain2359ΔARY, showing that the mutant strain is specifically disturbed inarylsulfatase activity.

The mutant strain can be used to make a lactase preparation atindustrial scale, virtually devoid of arylsulfatase activity.

1. A process for producing a dairy product which comprises adding alactase obtained from yeast to a dairy product comprising lactose,wherein said lactase comprises less than 30 units arylsulfatase activityper NLU of lactase activity.
 2. The process of claim 1 wherein the dairyproduct produced is free of off-flavours produced by arylsulfatase. 3.The process according to claim 2, wherein said dairy product contains analkyl phenol substituted with a sulfate group.
 4. The process accordingto claim 2, wherein the dairy product contains milk protein.
 5. Theprocess according to claim 2, wherein the dairy product is selected fromthe group consisting of milk and a fermented milk product.
 6. Theprocess according to claim 2, wherein the level of arylsulfatase in thedairy product during said treating is at most 500*10E3 arylsulfataseunits per liter of dairy product.
 7. A dairy product obtainable by theprocess according to claim
 2. 8. The process according to claim 4,wherein the milk protein is selected from the group consisting of caseinand whey protein.
 9. The process according to claim 5, wherein thefermented milk product is yoghurt, whey or a hydrolysate.
 10. Theprocess according to claim 6, wherein the level of arylsulfatase in thedairy product during said treating is at most 250*10E3 arylsulfataseunits per liter of dairy product.
 11. The process according to claim 10,wherein the level of arylsulfatase in the dairy product during saidtreating is at most 100*10E3 arylsulfatase units per liter of dairyproduct.
 12. The process according to claim 10, wherein the level ofarylsulfatase in the dairy product during said treating is at most50*10E3 arylsulfatase units per liter of dairy product.
 13. The processaccording to claim 10, wherein the level of arylsulfatase in the dairyproduct during said treating is at most 25*10E3 arylsulfatase units perliter of dairy product.
 14. The process of claim 2 wherein said lactaseis obtained by: (a) separation of arylsulfatase using chromatographyfrom a preparation which comprises lactase and arylsulfatase, (b)cultivating a lactase expressing cell in growth medium to which sulphatehas been added to repress arylsulfatase expression, (c) using a lactaseexpressing cell in which the gene for arylsulfatase is eliminated ordisrupted; or (d) using a lactase expressing strain having less than 10%of the detectable intracellular or extracellular arylsulfatase activityof the wild type strain from which the lactase expressing strainoriginates.
 15. The process according to claim 14, wherein said lactaseis neutral lactase.
 16. The process according to claim 14, wherein saidlactase is neutral lactase from Kluyveromyces lactis.
 17. The processaccording to claim 14, wherein said lactase is a neutral lactase derivedfrom the cytoplasm of yeast.
 18. The process according to claim 14,wherein said off flavor is medicinal.
 19. The process according to claim14, wherein said lactase expressing strain has less than 5% of thedetectable intracellular or extracellular arylsulfatase activity of thewild type strain from which the lactase expressing strain originates.20. The process according to claim 14, wherein said lactase expressingstrain has less than 1% of the detectable intracellular or extracellulararylsulfatase activity of the wild type strain from which the lactaseexpressing strain originates.
 21. The process according to claim 14,wherein arylsulfatase activity is undetectable in said lactaseexpressing strain.
 22. The process of claim 1 wherein the yeast isKluyveromyces.
 23. The process of claim 22 wherein the yeast isKluyveromyces lactis.
 24. The process according to claim 14(c), whereinthe gene disruption is achieved by a method selected from one-step genedisruption, marker insertion, site directed mutagenesis, deletion, RNAinterference, or anti-sense RNA.
 25. The process according to claim14(c), wherein the lactase expressing cell in which the gene forarylsulfatase is eliminated or disrupted produces more lactase than thewild type cell under the same culture conditions.
 26. Lactase whichcomprises less than 30 units arylsulfatase activity per NLU of lactaseactivity.
 27. Dairy product comprising lactase of claim
 26. 28. Processto purify a lactase containing enzyme preparation which comprisesseparation of aryl-sulfatase from lactase using chromotagraphy. 29.Process comprising treating a substrate with an enzyme preparation,wherein the enzyme preparation is substantially free from arylsulfatase.30. Process for preparing an enzyme preparation, said process comprisingpurifying a crude enzyme preparation which contains an enzyme ofinterest and arylsulfatase, wherein arylsulfatase is separated from theenzyme of interest.
 31. Process to produce a host cell which is anarylsulfatase deficient strain, which comprises bringing a culture whichproduces arylsulfatase under conditions that part of the culture ismodified to form the host cell which is arylsulfatase deficient andisolating the host cell.
 32. A process to produce a polypeptide by amethod comprising: (a) cultivating an arylsulfatase deficient host cellin a nutrient medium, under conditions conductive to expression of thepolypeptide (b) expressing the polypeptide in said host cell, and (c)optionally recovering the polypeptide from the nutrient medium or fromthe host cell.
 33. Enzyme preparation comprising a lactase, which enzymepreparation comprises less than 30 units arylsulfatase activity per NLUof lactase activity.